Modified double-stranded RNA agents

ABSTRACT

One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The sense strand of the dsRNA agent comprises at least one thermally destabilizing nucleotide, and at least one said thermally destabilizing nucleotide occurring at a site opposite to the seed region (positions 2-8) of the antisense strand; and the antisense strand of the dsRNA agent comprises at least two modified nucleotides that provide the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification, wherein said modified nucleotides are separated by 11 nucleotides in length. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

This application is a Continuation of U.S. patent application Ser. No.16/384,644, filed Apr. 15, 2019, which is a Continuation of U.S. patentapplication Ser. No. 16/272,721, filed Feb. 11, 2019, which is aContinuation of U.S. patent application Ser. No. 15/504,855, filed Feb.17, 2017, now U.S. Pat. No. 10,233,448, which is a 371 National Stageapplication of International PCT Application No. PCT/US2015/045407,filed Aug. 14, 2015, and claims benefit of priority to U.S. ProvisionalApplication No. 62/093,919, filed Dec. 18, 2014, U.S. ProvisionalApplication No. 62/083,744, filed Nov. 24, 2014, and U.S. ProvisionalApplication No. 62/039,507, filed Aug. 20, 2014, all of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to RNAi duplex agents having particular motifsthat are advantageous for inhibition of target gene expression, as wellas RNAi compositions suitable for therapeutic use. Additionally, theinvention provides methods of inhibiting the expression of a target geneby administering these RNAi duplex agents, e.g., for the treatment ofvarious diseases.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNAi (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

Double-stranded RNA (dsRNA) molecules with good gene-silencingproperties are needed for drug development based on RNA interference(RNAi). An initial step in RNAi is the activation of the RNA inducedsilencing complex (RISC), which requires degradation of the sense strandof the dsRNA duplex. Sense strand was known to act as the first RISCsubstrate that is cleaved by Argonaute 2 in the middle of the duplexregion. Immediately after the cleaved 5′-end and 3′-end fragments of thesense strand are removed from the endonuclease Ago2, the RISC becomesactivated by the antisense strand (Rand et al. (2005) Cell 123, 621).

It was believed that when the cleavage of the sense strand is inhibited,the endonucleolytic cleavage of target mRNA is impaired (Leuschner etal. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarzet al. (2004) Curr. Biol. 14, 787). Leuschner et al. showed thatincorporation of a 2′-O-Me ribose to the Ago2 cleavage site in the sensestrand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep.,7, 314). A similar effect was observed with phosphorothioatemodifications, showing that cleavage of the sense strand was requiredfor efficient RNAi also in mammals.

Morrissey et al. used a siRNA duplex containing 2′-F modified residues,among other sites and modifications, also at the Ago2 cleavage site, andobtained compatible silencing compared to the unmodified siRNAs(Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey'smodification is not motif specific, e.g., one modification includes 2′-Fmodifications on all pyrimidines on both sense and antisense strands aslong as pyrimidine residue is present, without any selectivity; andhence it is uncertain, based on these teachings, if specific motifmodification at the cleavage site of sense strand can have any actualeffect on gene silencing activity.

Muhonen et al. used a siRNA duplex containing two 2′-F modified residuesat the Ago2 cleavage site on the sense or antisense strand and found itwas tolerated (Muhonen et al. (2007) Chemistry & Biodiversity 4,858-873). However, Muhonen's modification is also sequence specific,e.g., for each particular strand, Muhonen only modifies either allpyrimidines or all purines, without any selectivity.

Choung et al. used a siRNA duplex containing alternative modificationsby 2′-OMe or various combinations of 2′-F, 2′-OMe and phosphorothioatemodifications to stabilize siRNA in serum to Sur10058 (Choung et al.(2006) Biochemical and Biophysical Research Communications 342,919-927). Choung suggested that the residues at the cleavage site of theantisense strand should not be modified with 2′-OMe in order to increasethe stability of the siRNA.

There is thus an ongoing need for iRNA duplex agents to improve the genesilencing efficacy of siRNA gene therapeutics. This invention isdirected to that need.

SUMMARY

This invention provides effective nucleotide or chemical motifs fordsRNA agents optionally conjugated to at least one ligand, which areadvantageous for inhibition of target gene expression, as well as RNAicompositions suitable for therapeutic use.

In one aspect, the invention relates to a double-stranded RNA (dsRNA)agent for inhibiting the expression of a target gene. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 40 nucleotides. The dsRNA agent is represented by formula (I):

In formula (I), B1, B2, B3, B1′, B2′, B3′, and B4′ each areindependently a nucleotide containing a modification selected from thegroup consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substitutedalkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′,B2′, B3′, and B4′ each contain 2′-OMe modifications. In one embodiment,B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-Fmodifications. In one embodiment, at least one of B1, B2, B3, B1′, B2′,B3′, and B4′ contain 2′-O—N-methylacetamido (2′-O-NMA) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite tothe seed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). For example, C1 is at a position of thesense strand that pairs with a nucleotide at positions 2-8 of the 5′-endof the antisense strand. In one example, C1 is at position 15 from the5′-end of the sense strand. C1 nucleotide bears the thermallydestabilizing modification which can include abasic modification;mismatch with the opposing nucleotide in the duplex; and sugarmodification such as 2′-deoxy modification or acyclic nucleotide e.g.,unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In oneembodiment, C1 has thermally destabilizing modification selected fromthe group consisting of: i) mismatch with the opposing nucleotide in theantisense strand; ii) abasic modification selected from the groupconsisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, le and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, thethermally destabilizing modification in C1 is a mismatch selected fromthe group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T,U:U, T:T, and U:T; and optionally, at least one nucleobase in themismatch pair is a 2′-deoxy nucleobase. In one example, the thermallydestabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a modification providing the nucleotide a steric bulk that isless or equal to the steric bulk of a 2′-OMe modification. A steric bulkrefers to the sum of steric effects of a modification. Methods fordetermining steric effects of a modification of a nucleotide are knownto one skilled in the art. The modification can be at the 2′ position ofa ribose sugar of the nucleotide, or a modification to a non-ribosenucleotide, acyclic nucleotide, or the backbone of the nucleotide thatis similar or equivalent to the 2′ position of the ribose sugar, andprovides the nucleotide a steric bulk that is less than or equal to thesteric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′are each independently selected from DNA, RNA, LNA, 2′-F, and2′-F-5′-methyl. In one embodiment, T1 is DNA. In one embodiment, T1′ isDNA, RNA or LNA. In one embodiment, T2′ is DNA or RNA. In oneembodiment, T3′ is DNA or RNA.

n¹, n³, and q¹ are independently 4 to 15 nucleotides in length.

n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length.

n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length;alternatively, n⁴ is 0.

q⁵ is independently 0-10 nucleotide(s) in length.

n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with twophosphorothioate internucleotide linkage modifications within position1-5 of the sense strand (counting from the 5′-end of the sense strand),and two phosphorothioate internucleotide linkage modifications atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴, q², q⁴, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n⁴is 1. In one embodiment, C1 is at position 15 of the 5′-end of the sensestrand

In one embodiment, T3′ starts at position 2 from the 5′ end of theantisense strand. In one example, T3′ is at position 2 from the 5′ endof the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ endof the antisense strand and T1′ starts from position 14 from the 5′ endof the antisense strand. In one example, T3′ starts from position 2 fromthe 5′ end of the antisense strand and q⁶ is equal to 1 and T1′ startsfrom position 14 from the 5′ end of the antisense strand and q² is equalto 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length(i.e. not counting the T1′ and T3′ nucleotides).

In one embodiment, T1′ is at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1, and the modification atthe 2′ position or positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisensestrand. In one example, T3′ is at position 2 from the 5′ end of theantisense strand and q⁶ is equal to 1, and the modification at the 2′position or positions in a non-ribose, acyclic or backbone that provideless than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at the cleavage site of the sense strand. Inone example, T1 is at position 11 from the 5′ end of the sense strand,when the sense strand is 19-22 nucleotides in length, and n² is 1. In anexemplary embodiment, T1 is at the cleavage site of the sense strand atposition 11 from the 5′ end of the sense strand, when the sense strandis 19-22 nucleotides in length, and n² is 1,

In one embodiment, T2′ starts at position 6 from the 5′ end of theantisense strand. In one example, T2′ is at positions 6-10 from the 5′end of the antisense strand, and q⁴ is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sensestrand, for instance, at position 11 from the 5′ end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n² is1; T1′ is at position 14 from the 5′ end of the antisense strand, and q²is equal to 1, and the modification to T1′ is at the 2′ position of aribose sugar or at positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10from the 5′ end of the antisense strand, and q⁴ is 1; and T3′ is atposition 2 from the 5′ end of the antisense strand, and q⁶ is equal to1, and the modification to T3′ is at the 2′ position or at positions ina non-ribose, acyclic or backbone that provide less than or equal tosteric bulk than a 2′-OMe ribose.

In one embodiment, T2′ starts at position 8 from the 5′ end of theantisense strand. In one example, T2′ starts at position 8 from the 5′end of the antisense strand, and q⁴ is 2.

In one embodiment, T2′ starts at position 9 from the 5′ end of theantisense strand. In one example, T2′ is at position 9 from the 5′ endof the antisense strand, and q⁴ is 1.

In one embodiment, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1,B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; withtwo phosphorothioate internucleotide linkage modifications withinpositions 1-5 of the sense strand (counting from the 5′-end of the sensestrand), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT atthe 3′-end of the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT atthe 3′-end of the antisense strand; with two phosphorothioateinternucleotide linkage modifications within positions 1-5 of the sensestrand (counting from the 5′-end of the sense strand), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the 5′-endof the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end). In one embodiment, B1 is2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F,q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

The dsRNA agent can comprise a phosphorus-containing group at the 5′-endof the sense strand or antisense strand. The 5′-endphosphorus-containing group can be 5′-end phosphate (5′-P), 5′-endphosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-endvinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or5′-deoxy-5′-C-malonyl

When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate(5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e.,trans-vinylphosphate,

5′-Z-VP isomer (i.e., cis-vinylphosphate,

or mixtures thereof.

In one embodiment, the dsRNA agent comprises a phosphorus-containinggroup at the 5′-end of the sense strand. In one embodiment, the dsRNAagent comprises a phosphorus-containing group at the 5′-end of theantisense strand.

In one embodiment, the dsRNA agent comprises a 5′-P. In one embodiment,the dsRNA agent comprises a 5′-P in the antisense strand.

In one embodiment, the dsRNA agent comprises a 5′-PS. In one embodiment,the dsRNA agent comprises a 5′-PS in the antisense strand.

In one embodiment, the dsRNA agent comprises a 5′-VP. In one embodiment,the dsRNA agent comprises a 5′-VP in the antisense strand. In oneembodiment, the dsRNA agent comprises a 5′-E-VP in the antisense strand.In one embodiment, the dsRNA agent comprises a 5′-Z-VP in the antisensestrand.

In one embodiment, the dsRNA agent comprises a 5′-PS₂. In oneembodiment, the dsRNA agent comprises a 5′-PS₂ in the antisense strand.

In one embodiment, the dsRNA agent comprises a 5′-PS₂. In oneembodiment, the dsRNA agent comprises a 5′-deoxy-5′-C-malonyl in theantisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-VP. The 5′-VPmay be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-VP. The 5′-VP maybe 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNA agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1. The dsRNA agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The dsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The dsRNA agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The dsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1. The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP,or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35% or 30% of the dsRNA agent of the invention ismodified. For example, when 50% of the dsRNA agent is modified, 50% ofall nucleotides present in the dsRNA agent contain a modification asdescribed herein.

In one embodiment, each of the sense and antisense strands of the dsRNAagent is independently modified with acyclic nucleotides, LNA, HNA,CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-fluoro, 2′-O—N-methylacetamido (2′-O-NMA), a2′-0-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP),or 2′-ara-F.

In one embodiment, each of the sense and antisense strands of the dsRNAagent contains at least two different modifications.

In one embodiment, the dsRNA agent of Formula (I) further comprises 3′and/or 5′ overhang(s) of 1-10 nucleotides in length. In one example,dsRNA agent of formula (I) comprises a 3′ overhang at the 3′-end of theantisense strand and a blunt end at the 5′-end of the antisense strand.In another example, the dsRNA agent has a 5′ overhang at the 5′-end ofthe sense strand.

In one embodiment, the dsRNA agent of the invention does not contain any2′-F modification.

In one embodiment, the sense strand and/or antisense strand of the dsRNAagent comprises one or more blocks of phosphorothioate ormethylphosphonate internucleotide linkages. In one example, the sensestrand comprises one block of two phosphorothioate or methylphosphonateinternucleotide linkages. In one example, the antisense strand comprisestwo blocks of two phosphorothioate or methylphosphonate internucleotidelinkages. For example, the two blocks of phosphorothioate ormethylphosphonate internucleotide linkages are separated by 16-18phosphate internucleotide linkages.

In one embodiment, each of the sense and antisense strands of the dsRNAagent has 15-30 nucleotides. In one example, the sense strand has 19-22nucleotides, and the antisense strand has 19-25 nucleotides. In anotherexample, the sense strand has 21 nucleotides, and the antisense strandhas 23 nucleotides.

In one embodiment, the nucleotide at position 1 of the 5′-end of theantisense strand in the duplex is selected from the group consisting ofA, dA, dU, U, and dT. In one embodiment, at least one of the first,second, and third base pair from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the antisense strand of the dsRNA agent of theinvention is 100% complementary to a target RNA to hybridize thereto andinhibits its expression through RNA interference. In another embodiment,the antisense strand of the dsRNA agent of the invention is at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, or at least 50%complementary to a target RNA.

In one aspect, the invention relates to a dsRNA agent as defined hereincapable of inhibiting the expression of a target gene. The dsRNA agentcomprises a sense strand and an antisense strand, each strand having 14to 40 nucleotides. The sense strand contains at least one thermallydestabilizing nucleotide, wherein at least one of said thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand). Each of the embodiments and aspectsdescribed in this specification relating to the dsRNA represented byformula (I) can also apply to the dsRNA containing the thermallydestabilizing nucleotide.

The thermally destabilizing nucleotide can occur, for example, betweenpositions 14-17 of the 5′-end of the sense strand when the sense strandis 21 nucleotides in length. The antisense strand contains at least twomodified nucleic acids that are smaller than a sterically demanding2′-OMe modification. Preferably, the two modified nucleic acids that aresmaller than a sterically demanding 2′-OMe are separated by 11nucleotides in length. For example, the two modified nucleic acids areat positions 2 and 14 of the 5′ end of the antisense strand. In oneembodiment, the dsRNA agent further comprises at least one ASGPR ligand.For example, the ASGPR ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker, such as:

In one example, the ASGPR ligand is attached to the 3′ end of the sensestrand.

For example, the dsRNA agent as defined herein can comprise i) aphosphorus-containing group at the 5′-end of the sense strand orantisense strand; ii) with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand); andiii) a ligand, such as a ASGPR ligand (e.g., one or more GalNAcderivatives) at 5′-end or 3′-end of the sense strand or antisensestrand. For instance, the ligand may be at the 3′-end of the sensestrand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-P and a targeting ligand.In one embodiment, the 5′-P is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof), and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-P and a targetingligand. In one embodiment, the 5′-P is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-PS and a targetingligand. In one embodiment, the 5′-PS is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-VP (e.g., a5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-PS₂ and atargeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of theantisense strand, and the targeting ligand is at the 3′-end of the sensestrand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyland a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl isat the 5′-end of the antisense strand, and the targeting ligand is atthe 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-P and a targeting ligand.In one embodiment, the 5′-P is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The dsRNA agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-P and a targeting ligand. In oneembodiment, the 5′-P is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-PS and a targeting ligand. In oneembodiment, the 5′-PS is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, orcombination thereof) and a targeting ligand. In one embodiment, the5′-VP is at the 5′-end of the antisense strand, and the targeting ligandis at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-PS₂ and a targeting ligand. In oneembodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). ThedsRNA agent also comprises a 5′-deoxy-5′-C-malonyl and a targetingligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end ofthe antisense strand, and the targeting ligand is at the 3′-end of thesense strand.

In a particular embodiment, the dsRNA agents of the present inventioncomprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker; and    -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, 13,        17, 19, and 21, and 2′-OMe modifications at positions 2, 4, 6,        8, 12, 14 to 16, 18, and 20 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15,        17, 19, 21, and 23, and 2′F modifications at positions 2, 4, 6        to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 21 and 22, and between nucleotide positions        22 and 23 (counting from the 5′ end);    -   wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, 13,        15, 17, 19, and 21, and 2′-OMe modifications at positions 2, 4,        6, 8, 12, 14, 16, 18, and 20 (counting from the 5′ end); and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13,        15, 17, 19, and 21 to 23, and 2′F modifications at positions 2,        4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and 12 to        21, 2′-F modifications at positions 7, and 9, and a        desoxy-nucleotide (e.g. dT) at position 11 (counting from the 5′        end); and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15,        17, and 19 to 23, and 2′-F modifications at positions 2, 4 to 6,        8, 10, 12, 14, 16, and 18 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12, 14,        and 16 to 21, and 2′-F modifications at positions 7, 9, 11, 13,        and 15; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15,        17, 19, and 21 to 23, and 2′-F modifications at positions 2 to        4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5′ end);        and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 9, and 12 to 21,        and 2′-F modifications at positions 10, and 11; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13,        15, 17, 19, and 21 to 23, and 2′-F modifications at positions 2,        4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11, and        13, and 2′-OMe modifications at positions 2, 4, 6, 8, 12, and 14        to 21; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to        13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at        positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′        end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14,        15, 17, and 19 to 21, and 2′-F modifications at positions 3, 5,        7, 9 to 11, 13, 16, and 18; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 25 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13,        15, 17, and 19 to 23, 2′-F modifications at positions 2, 3, 5,        8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g. dT) at        positions 24 and 25 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a four nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to 21,        and 2′-F modifications at positions 7, and 9 to 11; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to        13, 15, and 17 to 23, and 2′-F modifications at positions 2, 6,        9, 14, and 16 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to 21,        and 2′-F modifications at positions 7, and 9 to 11; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 23 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13,        15, and 17 to 23, and 2′-F modifications at positions 2, 6, 8,        9, 14, and 16 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 21 and 22, and between        nucleotide positions 22 and 23 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, the dsRNA agents of the presentinvention comprise:

(a) a sense strand having:

-   -   (i) a length of 19 nucleotides;    -   (ii) an ASGPR ligand attached to the 3′-end, wherein said ASGPR        ligand comprises three GalNAc derivatives attached through a        trivalent branched linker;    -   (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to 19,        and 2′-F modifications at positions 5, and 7 to 9; and    -   (iv) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, and between nucleotide positions 2        and 3 (counting from the 5′ end);

and

(b) an antisense strand having:

-   -   (i) a length of 21 nucleotides;    -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13,        15, and 17 to 21, and 2′-F modifications at positions 2, 6, 8,        9, 14, and 16 (counting from the 5′ end); and    -   (iii) phosphorothioate internucleotide linkages between        nucleotide positions 1 and 2, between nucleotide positions 2 and        3, between nucleotide positions 19 and 20, and between        nucleotide positions 20 and 21 (counting from the 5′ end);        wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In one embodiment, the dsRNA agents described herein further comprise athermally destabilizing modification at position 7 counting from the5′-end of the antisense from, at position 15 counting from the 5′-end ofsense strand, position 21 counting from the 5′-end of the sense strand,or combinations thereof.

In one aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 40nucleotides. The sense strand contains at least one thermallydestabilizing nucleotide, wherein at least one said thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand when the sense strand is 21 nucleotides in length. Theantisense strand comprises two modified nucleic acids that are smallerthan a sterically demanding 2′-OMe modification separated by 11nucleotides in length. For example, the two modified nucleic acids areat positions 2 and 14 of the 5′ end of the antisense strand.

In one embodiment, the sense strand of the dsRNA agent further comprisesan endonuclease susceptible modified nucleotide at the cleavage site ofthe sense strand. In one example, the endonuclease susceptible modifiednucleotide is at position 11 from the 5′ end of the sense strand.

In one embodiment, the antisense strand further comprises a thirdmodified nucleotide that provides the nucleotide a steric bulk that isless or equal to the steric bulk of a 2′-OMe modification, and the thirdmodified nucleotide is at positions 6-10 from the 5′ end of theantisense strand. For example, the third modified nucleotide is atposition 10 from the 5′ end of the antisense strand.

The embodiments for the thermally destabilizing nucleotides are similaras the various embodiments described above for C1 in formula (I). Theembodiments for the modified nucleic acids smaller than a stericallydemanding 2′-OMe modification are similar as the various embodimentsdescribed above for T1′, T2′, and T3′ in formula (I). The embodimentsdescribing the lengths, overhangs, additional modifications, and ligandconjugations to the dsRNA agents of formula I above are suitable here.

The present invention further relates to a use of a dsRNA agent asdefined herein for inhibiting expression of a target gene. In oneembodiment, the present invention further relates to a use of a dsRNAagent for inhibiting expression of a target gene in vitro.

The present invention further relates to a dsRNA agent as defined hereinfor use in inhibiting expression of a target gene in a subject. Thesubject may be any animal, preferably a mammal, more preferably a mouse,a rat, a sheep, a cattle, a dog, a cat, or a human.

In one aspect, the invention relates to a dsRNA agent capable ofinhibiting the expression of a target gene. The dsRNA agent comprises asense strand and an antisense strand, each strand having 14 to 40nucleotides. The sense strand comprises an endonuclease susceptiblemodified nucleotide (e.g., DNA, RNA, or 2′-F) near the cleavage site ofthe sense strand. For example, the endonuclease susceptible modifiednucleotide is at position 11 from the 5′ end of the sense strand. Theendonuclease susceptible modification that occurs near the cleavage sitecan influence the susceptibility of the cleavage site. For example,thermally destabilizing modifications near the cleavage site can provideendonuclease susceptibility at the cleavage site. The antisense strandcomprises two modified nucleic acids that are smaller than a stericallydemanding 2′-OMe modification separated by 11 nucleotides in length. Forexample, the two modified nucleic acids are at positions 2 and 14 of the5′ end of the antisense strand.

In another aspect, the invention further provides a method fordelivering the dsRNA agent of the invention to a specific target in asubject by subcutaneous or intravenous administration. The inventionfurther provides the dsRNA agents of the invention for use in a methodfor delivering said agents to a specific target in a subject bysubcutaneous or intravenous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows charts showing the effect of different modifications atsense strand position 17 on in vitro efficacy evaluated at 10 nM and 0.1nM concentration: (A) siRNAs targeting mTTR with non-F sense strandpaired with parent AS-strand; (B) siRNAs targeting mTTR with non-F sensestrand paired with non-F AS-strand; (C) siRNAs targeting ANG, ApoC3 andTTRSC with non-F sense strand paired with parent AS-strand.

FIG. 2 is a chart showing positional effect on in vitro efficacy ofthermally destabilizing GNA modification across the sense strandpositions 16-18 evaluated at 10 nM and 0.1 nM concentrations.

FIG. 3 is a chart showing the effect of modifications at the antisensestrand position 2 on in vitro efficacy of siRNAs targeting mTTR, ApoC3,TTRSC and TMP evaluated at 10 nM and 0.1 nM concentrations.

FIG. 4 is a chart showing the effect of modifications at the antisensestand position 14 on in vitro efficacy of siRNAs targeting mTTR, ApoC3,and TTRSC evaluated at 10 nM and 0.1 nM concentrations.

FIG. 5 is a graph showing the mTTR silencing in mice after a single SCdose of 2.5 mg/kg.

FIG. 6 is a chart showing the dose response of non-F siRNAs AD-61398 andAD-64273 compared to parent 2PS (AD-43527) and 6 PS (AD-57727); singleSC dose, protein levels measured 96 h post dose.

FIG. 7 is a chart showing the mTTR protein reduction in plasma after QWSC dosing of 1 mg/kg siRNA in mice comparing non-F AD-61398 with parentmotif: AD-57727.

FIG. 8 is a chart showing the silencing of TMPRSS6 mRNA after single SCdose of 3 mg/kg in mice (n=3/group): comparison of non-F designs withparent motif: AD-60490.

FIG. 9 is a graph showing the silencing of TMPRSS6 mRNA 7 days aftersingle SC dose of 3 mg/kg in mice (n=3/group): comparison of non-Fdesigns with parent motif: AD-60490.

FIG. 10 shows the in vitro activity results of two motifs, Motif 1 andMotif 2, activity compared to the parent compound AD-57727.

FIG. 11 shows the in vivo evaluation of the silencing activity of thesiRNAs targeting mTTR.

FIG. 12 shows the enhanced activity with Stability Enhanced ConjugateChemistry (SEC-C), where the liver was assessed for activity (mRNA) onday 7 post-dosing.

FIG. 13 depicts a chart showing an approximate 4-fold improvement inactivity with new motifs (Motifs 1 and 2) compared to the parentcompound.

FIG. 14 depicts a chart showing a markedly improved duration of Motif 1and Motif 2 across three sequences.

FIG. 15 depicts a graph showing the results of ApoC3-GalNAc3 SAR, atsingle 3 mg/kg SC dose hAAV 1×10¹¹ GC/mouse.

FIG. 16 illustrates a schematic of Ago2 loaded siRNA and the5′-vinylphosphonate (5′-VP), a modified phosphate mimicking stablephosphate. The 5′-phosphonate is added by cytosolic Clp1 kinase and actsas critical anchor for Ago2 loading.

FIG. 17 depicts a chart showing how the presence of 5′-VP generallyimproves in vivo activity. The evaluations were carried out on fourdifferent ApoB sequences. The LDL levels 7 days post single SC dose of 3mg/kg were analyzed for the four conjugates (with or without 5′-VPmodification).

FIG. 18 depicts different chemical modifications that can replace the PSlinkage and provide more stable chemistries, includingphosphorodithioate (PS₂), and methylphosphonate (MePhos), which promotesendogenous phosphorylation.

FIG. 19 shows a chart of an in vitro evaluation of end modifications,including 2′-OMe-MePhos, 2′-OMe-PS, dN(PS₂), and 2′F-PS. Transfection ofprimary mouse hepatocytes at 10 nM and 0.1 nM (n=4) were carried out ontwo ApoB conjugates.

FIG. 20 shows two charts showing how a minor change at the antisense5′-end can significantly improve in vivo efficacy. The chart on theleft, A), shows that 2′F-PS at position 1 of the antisense strand canimprove activity of 5′P-dependent sequences (with a single 3 mg/kg SCdose, and F9 activity was measured at day 3). The chart on the right,B), shows a ˜3-fold improved potency by dN(PS)₂ over the parent, similarto VP (with a single 10 mg/kg SC dose, and LDL was measured at day 3 forApoB).

FIG. 21 shows the SAR analysis of in vitro and in vivo activity of ApoBsiRNAs containing 5′-OH versus 5′-E-VP modification (at the 5′-end ofthe antisense strand). A) shows the results with in vitro transfectionmouse 1° hepatocytes. B) shows the LDL levels 3 days post a singledosing (SC dosing).

FIG. 22 shows the results of in vitro potency of 5′-E-VP modificationversus 5′-Z-VP modification to mTTR and F9 siRNA-GalNAc conjugates. Theresults were from in vitro transfection mouse primary hepatocytes.

FIG. 23 shows the results of in vivo comparison of 5′-E-VP modificationversus 5′-Z-VP modification to F9 siRNA-GalNAc conjugate (single SCdosing).

FIG. 24 shows graphs showing the dose-response curves for (A) 5′-OH, (B)5′-C-malonyl, and (C) 5′-phosphate PTEN siRNAs in primary mousehepatocytes in an in vitro PTEN silencing assay. All values are fromtriplicate experiments.

FIG. 25 shows the results of enzymatic stabilities of 5′-OH,5′-C-malonyl, and 5′-phosphate siRNAs incubated in in rat livertritosomes. The siRNA target sequences are shown in Table 10. The datawere normalized to untreated controls.

FIG. 26 shows the results of RISC loading of 5′-OH, 5′-C-malonyl, and5′-phosphate siRNAs (5′-modification on the antisense strands)determined by immunoprecipitation of Ago2 from primary mouse hepatocytesand by RT-PCR amplification of the Ago2-loaded single strands. Levels ofendogenous miR122 were determined as a control. The siRNA targetsequences are shown in Table 10.

FIG. 27 is a graph showing the in vitro knockdown of TTR using siRNAmodified with a single (S)-GNA nucleotide. Levels of TTR mRNA weremeasured after incubation with 10 nM siRNA in primary mouse hepatocytesfor 24 hours. TTR mRNA was assessed using RT-qPCR and normalized to PBStreated cells. All data points were the average of four measurements.

In FIG. 28, A) is a graph showing the in vitro knockdown of TTR usingsiRNA modified with a single (S)-GNA base pair. Levels of TTR mRNA weremeasured after incubation with 10 nM siRNA in primary mouse hepatocytesfor 24 hours. TTR mRNA was assessed using RT-qPCR and normalized to PBStreated cells. All data points were the average of four measurements. B)shows mix and match duplexes where sense and antisense strandscontaining single (S)-GNA nucleotides were paired as GNA:RNA hetero-basepairs.

FIG. 29 is a graph showing the in vivo levels of TTR in mouse serum.Animals received a single dose of 2.5 mg/kg siRNA. At the indicated timepre- or post-dosing, animals were bled and serum samples were measuredusing a sandwich ELISA assay utilizing a HRP-conjugate antibody and3,3′,5,5′-tetramethylbenzidine for readout at 450 nm. All samples weremeasured in duplicate and each data point is the average of the mice ineach cohort (n=3).

FIG. 30 is a graph showing the in vivo quantification of TTR mRNAlevels. Animals received a single dose of 2.5 mg/kg siRNA. At theindicated time post-dosing, RNA extraction was performed on whole-liverhomogenate. TTR mRNA was assessed as above by RT-qPCR, using the ΔΔCtmethod with GAPDH as the control transcript, and normalized toPBS-treated animals. Dark bars indicate the results for Day 21; andblank bars indicate the results for Day 7.

DETAILED DESCRIPTION

The inventors found that having 2′-OMe modifications at nucleotidepositions 2 and 14 from the 5′-end of the antisense strand dampened thegene silencing activity of a dsRNA agent. By introducing chemicalmodifications at the 2′ position or equivalent positions in anon-ribose, acyclic or backbone that provide less steric bulk than a2′-OMe modification at certain positions in antisense and/or sensestrand, the dsRNA agents were able to regain the gene silencingactivity. The inventors also determined that introducing a thermallydestabilizing nucleotide on the sense strand at a site opposite to theseed region of the antisense strand (i.e. at position 2-8 of the 5′-endof the antisense strand) provides better gene silencing activity.

The sense strand and antisense strand of the dsRNA agent may becompletely modified. The dsRNA agent optionally conjugates with anasialoglycoprotein receptor (ASGPR) ligand, for instance on the sensestrand. The resulting dsRNA agents present effective in vivo genesilencing activity.

Accordingly, the invention provides a double-stranded RNAi (dsRNA) agentcapable of inhibiting the expression of a target gene. The dsRNA agentcomprises a sense strand and an antisense strand. Each strand of thedsRNA agent can range from 12-40 nucleotides in length. For example,each strand can be between 14-40 nucleotides in length, 17-37nucleotides in length, 25-37 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex dsRNA. Theduplex region of a dsRNA agent may be 12-40 nucleotide pairs in length.For example, the duplex region can be between 14-40 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 25-35 nucleotides in length,27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length,17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length,19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length,19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or21-23 nucleotide pairs in length. In another example, the duplex regionis selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27nucleotide pairs in length.

In one embodiment, the dsRNA agent of the invention comprises one ormore overhang regions and/or capping groups of dsRNA agent at the3′-end, or 5′-end or both ends of a strand. The overhang can be 1-10nucleotides in length, 1-6 nucleotides in length, for instance 2-6nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides inlength, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides inlength. The overhangs can be the result of one strand being longer thanthe other, or the result of two strands of the same length beingstaggered. The overhang can form a mismatch with the target mRNA or itcan be complementary to the gene sequences being targeted or can beother sequence. The first and second strands can also be joined, e.g.,by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the dsRNAagent of the invention can each independently be a modified orunmodified nucleotide including, but not limited to 2′-sugar modified,such as, 2-F 2′-Omethyl, thymidine (T),2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine(Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof. For example, TT can be an overhang sequence for either end oneither strand. The overhang can form a mismatch with the target mRNA orit can be complementary to the gene sequences being targeted or can beother sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the dsRNA agent of the invention may be phosphorylated. Insome embodiments, the overhang region contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The dsRNA agent of the invention may comprise only a single overhang,which can strengthen the interference activity of the dsRNA, withoutaffecting its overall stability. For example, the single-strandedoverhang is located at the 3′-terminal end of the sense strand or,alternatively, at the 3′-terminal end of the antisense strand. The dsRNAmay also have a blunt end, located at the 5′-end of the antisense strand(or the 3′-end of the sense strand) or vice versa. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. While not bound by theory, the asymmetric bluntend at the 5′-end of the antisense strand and 3′-end overhang of theantisense strand favor the guide strand loading into RISC process. Forexample the single overhang comprises at least two, three, four, five,six, seven, eight, nine, or ten nucleotides in length.

In one embodiment, the dsRNA agent of the invention may also have twoblunt ends, at both ends of the dsRNA duplex.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 19 nt in length, wherein the sense strand contains at leastone thermally destabilizing nucleotide, where at least one thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand. The antisense strand contains at least two modifiednucleic acids that is smaller than a sterically demanding 2′-OMe;preferably, the two modified nucleic acids that is smaller than asterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 20 nt in length, wherein the sense strand contains at leastone thermally destabilizing nucleotide, where at least one thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand. The antisense strand contains at least two modifiednucleic acids that is smaller than a sterically demanding 2′-OMe;preferably, the two modified nucleic acids that is smaller than asterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand.

In one embodiment, the dsRNA agent of the invention is a double endedbluntmer of 21 nt in length, wherein the sense strand contains at leastone thermally destabilizing nucleotide, where at least one thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand. The antisense strand contains at least two modifiednucleic acids that is smaller than a sterically demanding 2′-OMe;preferably, the two modified nucleic acids that is smaller than asterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand.

In one embodiment, the dsRNA agent of the invention comprises a 21nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense,wherein the sense strand contains at least one thermally destabilizingnucleotide, where at least one thermally destabilizing nucleotide occursat or near the site that is opposite to the seed region of the antisensestrand (i.e. at position 2-8 of the 5′-end of the antisense strand), Forexample, the thermally destabilizing nucleotide occurs between positions14-17 of the 5′-end of the sense strand when the sense strand is 21nucleotides in length. The antisense strand contains at least twomodified nucleic acids that is smaller than a sterically demanding2′-OMe; preferably, the two modified nucleic acids that is smaller thana sterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand, wherein one end of the dsRNA is blunt, while theother end is comprises a 2 nt overhang. Preferably, the 2 nt overhang isat the 3′-end of the antisense. Optionally, the dsRNA further comprisesa ligand (preferably a receptor ligand i.e. ASGPR ligand).

In one embodiment, the dsRNA agent of the invention comprising a senseand antisense strands, wherein: the sense strand is 25-30 nucleotideresidues in length, wherein starting from the 5′ terminal nucleotide(position 1), positions 1 to 23 of said sense strand comprise at least 8ribonucleotides; antisense strand is 36-66 nucleotide residues in lengthand, starting from the 3′ terminal nucleotide, at least 8ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when said double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least onethermally destabilizing nucleotide, where at least one thermallydestabilizing nucleotide occurs at or near the site that is opposite tothe seed region of the antisense strand (i.e. at position 2-8 of the5′-end of the antisense strand), For example, the thermallydestabilizing nucleotide occurs between positions 14-17 of the 5′-end ofthe sense strand. The antisense strand contains at least two modifiednucleic acids that are smaller than a sterically demanding 2′-OMe;preferably, the two modified nucleic acids that are smaller than asterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand.

In one embodiment, the dsRNA agent of the invention comprises a senseand antisense strands, wherein said dsRNA agent comprises a sense strandhaving a length which is at least 25 and at most 29 nucleotides and anantisense strand having a length which is at most 30 nucleotides withthe sense strand comprises a modified nucleotide that is susceptible toenzymatic degradation at position 11 from the 5′ end. The antisensestrand comprises two modified nucleic acids that are smaller than asterically demanding 2′-OMe are at positions 2 and 14 of the 5′ end ofthe antisense strand; wherein said 3′ end of said sense strand and said5′ end of said antisense strand form a blunt end and said antisensestrand is 1-4 nucleotides longer at its 3′ end than the sense strand,wherein the duplex region which is at least 25 nucleotides in length,and said antisense strand is sufficiently complementary to a target mRNAalong at least 19 nt of said antisense strand length to reduce targetgene expression when said dsRNA agent is introduced into a mammaliancell, and wherein dicer cleavage of said dsRNA preferentially results inan siRNA comprising said 3′ end of said antisense strand, therebyreducing expression of the target gene in the mammal. Optionally, thedsRNA agent further comprises a ligand.

In one embodiment, the sense strand comprises a modified nucleotide thatis susceptible to enzymatic degradation at position 11 from the 5′ end.The antisense strand comprises two modified nucleic acids that aresmaller than a sterically demanding 2′-OMe are at positions 2 and 14 ofthe 5′ end of the antisense strand.

In one embodiment, the antisense strand comprises two modified nucleicacids that are smaller than a sterically demanding 2′-OMe are atpositions 2 and 14 of the 5′ end of the antisense strand.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the dsRNA agent may be modified. Each nucleotide may bemodified with the same or different modification which can include oneor more alteration of one or both of the non-linking phosphate oxygensand/or of one or more of the linking phosphate oxygens; alteration of aconstituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribosesugar; wholesale replacement of the phosphate moiety with “dephospho”linkers; modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. E.g., a phosphorothioate modification at a non-linking 0 positionmay only occur at one or both termini, may only occur in a terminalregion, e.g., at a position on a terminal nucleotide or in the last 2,3, 4, 5, or 10 nucleotides of a strand, or may occur in double strandand single strand regions, particularly at termini. The 5′ end or endscan be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. E.g., it can be desirable to include purine nucleotides inoverhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strandscan contain more than one modification. In one embodiment, each residueof the sense strand and antisense strand is independently modified with2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides orothers.

In one embodiment, the sense strand and antisense strand each comprisestwo differently modified nucleotides selected from 2′-O-methyl or2′-deoxy.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with 2′-O-methyl nucleotide, 2′-deoxynucleotide, 2′-deoxyfluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA)nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide,2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide.

In one embodiment, the dsRNA agent of the invention comprisesmodifications of an alternating pattern, particular in the B1, B2, B3,B1′, B2′, B3′, B4′ regions, as shown in formula I. The term “alternatingmotif” or “alternative pattern” as used herein refers to a motif havingone or more modifications, each modification occurring on alternatingnucleotides of one strand. The alternating nucleotide may refer to oneper every other nucleotide or one per every three nucleotides, or asimilar pattern. For example, if A, B and C each represent one type ofmodification to the nucleotide, the alternating motif can be“ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,”“AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,”etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the dsRNA agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 3′-5′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the anti sense strand may start with “BBAABBAA”from 3′-5′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

The dsRNA agent of the invention may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. Thephosphorothioate or methylphosphonate internucleotide linkagemodification may occur on any nucleotide of the sense strand orantisense strand or both in any position of the strand. For instance,the internucleotide linkage modification may occur on every nucleotideon the sense strand and/or antisense strand; each internucleotidelinkage modification may occur in an alternating pattern on the sensestrand or antisense strand; or the sense strand or antisense strandcomprises both internucleotide linkage modifications in an alternatingpattern. The alternating pattern of the internucleotide linkagemodification on the sense strand may be the same or different from theantisense strand, and the alternating pattern of the internucleotidelinkage modification on the sense strand may have a shift relative tothe alternating pattern of the internucleotide linkage modification onthe antisense strand.

In one embodiment, the dsRNA agent comprises the phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region comprises two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within duplex region. For example, at least 2, 3, 4, or allthe overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. Preferably, these terminal three nucleotides may be at the3′-end of the antisense strand.

In one embodiment, the sense strand of the dsRNA agent comprises 1-10blocks of two to ten phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one ofthe phosphorothioate or methylphosphonate internucleotide linkages isplaced at any position in the oligonucleotide sequence and the saidsense strand is paired with an antisense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of two phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said antisensestrand is paired with a sense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of three phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or 16 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said antisensestrand is paired with a sense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of four phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14phosphate internucleotide linkages, wherein one of the phosphorothioateor methylphosphonate internucleotide linkages is placed at any positionin the oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of five phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of six phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of seven phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotidelinkages, wherein one of the phosphorothioate or methylphosphonateinternucleotide linkages is placed at any position in theoligonucleotide sequence and the said antisense strand is paired with asense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of eight phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleotidelinkages, wherein one of the phosphorothioate or methylphosphonateinternucleotide linkages is placed at any position in theoligonucleotide sequence and the said antisense strand is paired with asense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In one embodiment, the antisense strand of the dsRNA agent comprises twoblocks of nine phosphorothioate or methylphosphonate internucleotidelinkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages,wherein one of the phosphorothioate or methylphosphonate internucleotidelinkages is placed at any position in the oligonucleotide sequence andthe said antisense strand is paired with a sense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In one embodiment, the dsRNA agent of the invention further comprisesone or more phosphorothioate or methylphosphonate internucleotidelinkage modification within 1-10 of the termini position(s) of the senseand/or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or10 nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage at one end or both ends of thesense and/or antisense strand.

In one embodiment, the dsRNA agent of the invention further comprisesone or more phosphorothioate or methylphosphonate internucleotidelinkage modification within 1-10 of the internal region of the duplex ofeach of the sense and/or antisense strand. For example, at least 2, 3,4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked throughphosphorothioate methylphosphonate internucleotide linkage at position8-16 of the duplex region counting from the 5′-end of the sense strand;the dsRNA agent can optionally further comprise one or morephosphorothioate or methylphosphonate internucleotide linkagemodification within 1-10 of the termini position(s).

In one embodiment, the dsRNA agent of the invention further comprisesone to five phosphorothioate or methylphosphonate internucleotidelinkage modification(s) within position 1-5 and one to fivephosphorothioate or methylphosphonate internucleotide linkagemodification(s) within position 18-23 of the sense strand (counting fromthe 5′-end), and one to five phosphorothioate or methylphosphonateinternucleotide linkage modification at positions 1 and 2 and one tofive within positions 18-23 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification withinposition 1-5 and one phosphorothioate or methylphosphonateinternucleotide linkage modification within position 18-23 of the sensestrand (counting from the 5′-end), and one phosphorothioateinternucleotide linkage modification at positions 1 and 2 and twophosphorothioate or methylphosphonate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and two phosphorothioate internucleotide linkagemodifications within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and two phosphorothioate internucleotide linkagemodifications within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification withinposition 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification withinposition 1-5 and one within position 18-23 of the sense strand (countingfrom the 5′-end), and two phosphorothioate internucleotide linkagemodification at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification withinposition 1-5 (counting from the 5′-end) of the sense strand, and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and one phosphorothioate internucleotide linkage modificationwithin positions 18-23 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 (counting from the 5′-end) of the sense strand, and onephosphorothioate internucleotide linkage modification at positions 1 and2 and two phosphorothioate internucleotide linkage modifications withinpositions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and one within position 18-23 of the sense strand (countingfrom the 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications withinposition 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications at position 1and 2, and two phosphorothioate internucleotide linkage modifications atposition 20 and 21 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one at position 21 of the antisense strand (counting from the5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification at position 1,and one phosphorothioate internucleotide linkage modification atposition 21 of the sense strand (counting from the 5′-end), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modifications atpositions 20 and 21 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications at position 1and 2, and two phosphorothioate internucleotide linkage modifications atposition 21 and 22 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one phosphorothioate internucleotide linkage modification atposition 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification at position 1,and one phosphorothioate internucleotide linkage modification atposition 21 of the sense strand (counting from the 5′-end), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modifications atpositions 21 and 22 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisestwo phosphorothioate internucleotide linkage modifications at position 1and 2, and two phosphorothioate internucleotide linkage modifications atposition 22 and 23 of the sense strand (counting from the 5′-end), andone phosphorothioate internucleotide linkage modification at positions 1and one phosphorothioate internucleotide linkage modification atposition 21 of the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention further comprisesone phosphorothioate internucleotide linkage modification at position 1,and one phosphorothioate internucleotide linkage modification atposition 21 of the sense strand (counting from the 5′-end), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modifications atpositions 23 and 23 the antisense strand (counting from the 5′-end).

In one embodiment, the dsRNA agent of the invention comprisesmismatch(es) with the target, within the duplex, or combinationsthereof. The mismatch can occur in the overhang region or the duplexregion. The base pair can be ranked on the basis of their propensity topromote dissociation or melting (e.g., on the free energy of associationor dissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:A:U is preferred over G:C; G:U is preferred over G:C; and I:C ispreferred over G:C (I=inosine). Mismatches, e.g., non-canonical or otherthan canonical pairings (as described elsewhere herein) are preferredover canonical (A:T, A:U, G:C) pairings; and pairings which include auniversal base are preferred over canonical pairings.

In one embodiment, the dsRNA agent of the invention comprises at leastone of the first 1, 2, 3, 4, or 5 base pairs within the duplex regionsfrom the 5′-end of the antisense strand can be chosen independently fromthe group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonicalor other than canonical pairings or pairings which include a universalbase, to promote the dissociation of the antisense strand at the 5′-endof the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

The inventors found that introducing 4′-modified and/or 5′-modifiednucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate(PS), and/or phosphorodithioate (PS2) linkage of a dinucleotide at anyposition of single stranded or double stranded oligonucleotide can exertsteric effect to the internucleotide linkage and, hence, protecting orstabilizing it against nucleases.

In one embodiment, 5′-modified nucleoside is introduced at the 3′-end ofa dinucleotide at any position of single stranded or double strandedsiRNA. For instance, a 5′-alkylated nucleoside may be introduced at the3′-end of a dinucleotide at any position of single stranded or doublestranded siRNA. The alkyl group at the 5′ position of the ribose sugarcan be racemic or chirally pure R or S isomer. An exemplary 5′-alkylatednucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemicor chirally pure R or S isomer.

In one embodiment, 4′-modified nucleoside is introduced at the 3′-end ofa dinucleotide at any position of single stranded or double strandedsiRNA. For instance, a 4′-alkylated nucleoside may be introduced at the3′-end of a dinucleotide at any position of single stranded or doublestranded siRNA. The alkyl group at the 4′ position of the ribose sugarcan be racemic or chirally pure R or S isomer. An exemplary 4′-alkylatednucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemicor chirally pure R or S isomer. Alternatively, a 4′-O-alkylatednucleoside may be introduced at the 3′-end of a dinucleotide at anyposition of single stranded or double stranded siRNA. The 4′-O-alkyl ofthe ribose sugar can be racemic or chirally pure R or S isomer. Anexemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The4′-O-methyl can be either racemic or chirally pure R or S isomer.

In one embodiment, 5′-alkylated nucleoside is introduced at any positionon the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 5′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can beeither racemic or chirally pure R or S isomer.

In one embodiment, 4′-alkylated nucleoside is introduced at any positionon the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 4′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can beeither racemic or chirally pure R or S isomer.

In one embodiment, 4′-O-alkylated nucleoside is introduced at anyposition on the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 5′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl canbe either racemic or chirally pure R or S isomer.

In one embodiment, the sense strand sequence of the dsRNA agent isrepresented by formula (Is):

wherein:

B1, B2, and B3 each independently represent a nucleotide containing amodification selected from the group consisting of 2′-Oalkyl,2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA, and BNA/LNA;

C1 is a thermally destabilizing nucleotide (e.g., acyclic nucleotidesuch as UNA or GNA, mismatch, abasic, or DNA) placed at the opposite ofthe antisense seed region (i.e., positions 2-8 of the 5′-end of theantisense strand);

T1 represents a nucleotide comprising a chemical modification at the 2′position or equivalent position in a non-ribose, acyclic or backbonethat provide the nucleotide a less steric bulk than a 2′-OMemodification; for example, T1 is selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ or n³ is independently 4 to 15 nucleotides in length;

n⁵ is 1-6 nucleotide(s) in length;

n⁴ is 1-3 nucleotide(s) in length; alternatively n⁴ is 0, and

n² is 0-3 nucleotide(s) in length.

In one embodiment, the sense strand sequence having 19, 20, 21, or 22nucleotides in length of the dsRNA agent is represented by formula (Is):

wherein:

B1, B2, and B3 each independently represent a nucleotide containing amodification selected from the group consisting of 2′-Oalkyl,2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA, and BNA/LNA;

C1 is a thermally destabilizing nucleotide (e.g., acyclic nucleotidesuch as UNA or GNA, mismatch, abasic, or DNA) placed at the opposite ofthe antisense seed region (i.e., positions 2-8 of the 5′-end of theantisense strand);

T1 represents a nucleotide comprising a chemical modification selectedfrom the group consisting of DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ or n³ is independently 4 to 15 nucleotides in length;

n⁵ is 1-6 nucleotide(s) in length;

n⁴ is 1-3 nucleotide(s) in length; alternatively n⁴ is 0 and

n² is 0-3 nucleotide(s) in length.

In one embodiment, the dsRNA agent of formula (Is) further comprises 3′and/or 5′ overhang(s) of 1-10 nucleotides in length. In one example, thedsRNA agent of formula (Is) comprises a 5′ overhang.

In one embodiment, C1 comprises one thermally destabilizing nucleotideat position 14, 15, 16 or 17 from the 5′-end of the sense strand. Forexample, C1 is an acyclic nucleotide (e.g., UNA or GNA), mismatch,abasic, or DNA. In one specific example, C1 is a GNA.

In one embodiment, T1 comprises a DNA, RNA, LNA, 2′-F, or 2′-F-5′-methylat position 11 from the 5′-end of the sense strand.

In one embodiment, the dsRNA agent of the invention comprises a sensestrand (Is), wherein C1 is an acyclic nucleotide (e.g., UNA or GNA),mismatch, abasic, or DNA; and T1 comprises a DNA, RNA, LNA, 2′-F, or2′-F-5′-methyl at position 11 from the 5′-end of the sense strand.

In one embodiment, the antisense strand sequence of the dsRNA agent isrepresented by formula (Ia):

wherein:

B1′, B2′, B3′, and B4′ each independently represent a nucleotidecontaining a modification selected from the group consisting of2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

T1′, T2′, and T3′ each independently represent a nucleotide comprising achemical modification at the 2′ position or equivalent position in anon-ribose, acyclic or backbone that provide the nucleotide a lesssteric bulk than a 2′-OMe modification; for example, T1′, T2′, and T3′each are independently selected from the group consisting of DNA, RNA,LNA, 2′-F, and 2′-F-5′-methyl;

q¹ is independently 4 to 15 nucleotides in length;

q³ or q⁷ is independently 1-6 nucleotide(s) in length;

q² or q⁶ is independently 1-3 nucleotide(s) in length;

q⁴ is independently 0-3 nucleotide(s) in length; and

q⁵ is independently 0-10 nucleotide(s) in length.

In one embodiment, the antisense strand sequence having 19, 20, 21, 22,23, 24, or 25 nucleotides in length of the dsRNA agent is represented byformula (Ia):

wherein:

B1′, B2′, B3′, and B4′ each independently represent a nucleotidecontaining a modification selected from the group consisting of2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

T1′, T2′, and T3′ each independently represent a nucleotide comprising achemical modification selected from the group consisting of DNA, RNA,LNA, 2′-F, and 2′-F-5′-methyl;

q¹ is independently 4 to 15 nucleotides in length;

q³ or q⁷ is independently 1-6 nucleotide(s) in length;

q² or q⁶ is independently 1-3 nucleotide(s) in length;

q⁴ is independently 0-3 nucleotide(s) in length; and

q⁵ is independently 0-10 nucleotide(s) in length.

In one embodiment, dsRNA of formula (Ia) further comprises 3′ and/or 5′overhang(s) of 1-10 nucleotides in length. In one example, dsRNA offormula (Ia) comprises a 3′ overhang.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification selected from the group consistingof 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

C1 is an acyclic nucleotide (e.g., UNA or GNA);

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a chemical modification selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ n³, or q¹ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has 3′ and/or 5′ overhang(s) of 1-10 nucleotidesin length of the antisense and/or sense strand(s).

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification selected from the group consistingof 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

C1 is an acyclic nucleotide (e.g., UNA or GNA);

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a chemical modification selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ n³, or q¹ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 3′ overhang of 2 nucleotides in length atthe 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 15-30 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification 2′-OMe;

C1 is an acyclic nucleotide GNA;

T1, T1′, T2′, and T3′ each are independently DNA or RNA;

n³, or q¹ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 3′ overhang of 1-6 nucleotides in lengthat the 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 19-23 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a 2′-OMe modification;

C1 is an acyclic nucleotide GNA;

T1, T1′, T2′, and T3′ are independently DNA or RNA;

n¹ n³, or q³ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n², q⁴ or q⁵ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 3′ overhang of 2 nucleotides in length atthe 3′-end of the antisense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification selected from the group consistingof 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

C1 is an acyclic nucleotide (e.g., UNA or GNA);

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a chemical modification selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ n³, or q¹ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 5′ overhang of 1-10 nucleotides in lengthat the 5′-end of the sense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification selected from the group consistingof 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

C1 is an acyclic nucleotide (e.g., UNA or GNA);

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a chemical modification selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl;

n¹ n³, or q¹ is independently 4 to 15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 5′ overhang of 1-6 nucleotides in lengthat the 5′-end of the sense.

In one embodiment, the invention relates to a double-stranded RNA(dsRNA) agent for inhibiting the expression of a target gene. The dsRNAagent comprises a sense strand and an antisense strand, each strandhaving 14 to 40 nucleotides:

wherein:

B1, B2, B3, B1′, B2′, B3′, and B4′ each independently represent anucleotide containing a modification selected from the group consistingof 2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA;

C1 is an acyclic nucleotide (e.g., UNA or GNA);

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a chemical modification selected from the group consisting ofDNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl; n³, or q¹ is independently 4 to15 nucleotides in length;

n⁵, q³ or q⁷ is independently 1-6 nucleotide(s) in length;

n⁴, q² or q⁶ is independently 1-3 nucleotide(s) in length; alternativelyn⁴ is 0,

n² or q⁴ is independently 0-3 nucleotide(s) in length;

q⁵ is independently 0-10 nucleotide(s) in length; and

wherein the dsRNA agent has a 5′ overhang of 1-10 nucleotides in lengthat the 5′-end of the sense and a 3′ overhang of 1-10 nucleotides inlength at the 5′-end of the antisense strand.

Thermally Destabilizing Modifications.

The dsRNA agent can be optimized for RNA interference by increasing thepropensity of the dsRNA duplex to disassociate or melt (decreasing thefree energy of duplex association) by introducing a thermallydestabilizing modification in the sense strand at a site opposite to theseed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). This modification can increase thepropensity of the duplex to disassociate or melt in the seed region ofthe antisense strand.

The thermally destabilizing modifications can include abasicmodification; mismatch with the opposing nucleotide in the opposingstrand; and sugar modification such as 2′-deoxy modification or acyclicnucleotide, e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid(GNA).

Exemplified abasic modifications are:

Exemplified sugar modifications are:

The term “acyclic nucleotide” refers to any nucleotide having an acyclicribose sugar, for example, where any of bonds between the ribose carbons(e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent and/orat least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′ orO4′) are independently or in combination absent from the nucleotide. Insome embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, le and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refersto unlocked acyclic nucleic acid, wherein any of the bonds of the sugarhas been removed, forming an unlocked “sugar” residue. In one example,UNA also encompasses monomers with bonds between C1′-C4′ being removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar isremoved (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059(1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which arehereby incorporated by reference in their entirety). The acyclicderivative provides greater backbone flexibility without affecting theWatson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similarto DNA or RNA but differing in the composition of its “backbone” in thatis composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification can be mismatches (i.e.,noncomplementary base pairs) between the thermally destabilizingnucleotide and the opposing nucleotide in the opposite strand within thedsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T,A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Othermismatch base pairings known in the art are also amenable to the presentinvention. A mismatch can occur between nucleotides that are eithernaturally occurring nucleotides or modified nucleotides, i.e., themismatch base pairing can occur between the nucleobases from respectivenucleotides independent of the modifications on the ribose sugars of thenucleotides. In certain embodiments, the dsRNA agent contains at leastone nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase;e.g., the 2′-deoxy nucleobase is in the sense strand.

More examples of abasic nucleotide, acyclic nucleotide modifications(including UNA and GNA), and mismatch modifications have been describedin detail in WO 2011/133876, which is herein incorporated by referencein its entirety.

The thermally destabilizing modifications may also include universalbase with reduced or abolished capability to form hydrogen bonds withthe opposing bases, and phosphate modifications.

Nucleobase modifications with impaired or completely abolishedcapability to form hydrogen bonds with bases in the opposite strand havebeen evaluated for destabilization of the central region of the dsRNAduplex as described in WO 2010/0011895, which is herein incorporated byreference in its entirety. Exemplary nucleobase modifications are:

Exemplary phosphate modifications known to decrease the thermalstability of dsRNA duplexes compared to natural phosphodiester linkagesare:

In one embodiment, the dsRNA agent of the invention can comprise 2′-5′linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S). For example,the 2′-5′ linkages modifications can be used to promote nucleaseresistance or to inhibit binding of the sense to the antisense strand,or can be used at the 5′ end of the sense strand to avoid sense strandactivation by RISC.

In another embodiment, the dsRNA agent of the invention can comprise Lsugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). Forexample, these L sugars modifications can be used to promote nucleaseresistance or to inhibit binding of the sense to the antisense strand,or can be used at the 5′ end of the sense strand to avoid sense strandactivation by RISC.

In one embodiment, the dsRNA agent is a multimer containing at least twoduplexes represented by formula (I), wherein said duplexes are connectedby a linker. The linker can be cleavable or non-cleavable. Optionally,said multimer further comprise a ligand. Each of the dsRNA agent cantarget the same gene or two different genes; or each of the dsRNA agentcan target same gene at two different target sites.

In one embodiment, the dsRNA agent is a multimer containing three, four,five, six or more duplexes represented by formula (I), wherein saidduplexes are connected by a linker. The linker can be cleavable ornon-cleavable. Optionally, said multimer further comprises a ligand.Each of the dsRNA agent can target the same gene or two different genes;or each of the dsRNA agent can target same gene at two different targetsites.

In one embodiment, two dsRNA agent represented by formula (I) are linkedto each other at the 5′ end, and one or both of the 3′ ends of the areoptionally conjugated to a ligand. Each of the dsRNA can target the samegene or two different genes; or each of the dsRNA can target same geneat two different target sites.

Various publications described multimeric siRNA and can all be used withthe dsRNA of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 which are hereby incorporated by their entirely.

The dsRNA agent that contains conjugations of one or more carbohydratemoieties to a dsRNA agent can optimize one or more properties of thedsRNA agent. In many cases, the carbohydrate moiety will be attached toa modified subunit of the dsRNA agent. E.g., the ribose sugar of one ormore ribonucleotide subunits of a dsRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrier towhich is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In one embodiment the dsRNA agent of the invention is conjugated to aligand via a carrier, wherein the carrier can be cyclic group or acyclicgroup; preferably, the cyclic group is selected from pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and decalin; preferably, the acyclic group is selectedfrom serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the invention may optionally beconjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand, in particular, the 3′-end of the sense strand.

In one embodiment dsRNA agents of the invention are 5′ phosphorylated orinclude a phosphoryl analog at the 5′ prime terminus. 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substitutedvinyl), (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). In one example, the modification can in placed in theantisense strand of a dsRNA agent.

Ligands

A wide variety of entities can be coupled to the oligonucleotides of thepresent invention. Preferred moieties are ligands, which are coupled,preferably covalently, either directly or indirectly via an interveningtether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, receptor e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Ligands providingenhanced affinity for a selected target are also termed targetingligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In one embodiment, the endosomolytic ligand assumes itsactive conformation at endosomal pH. The “active” conformation is thatconformation in which the endosomolytic ligand promotes lysis of theendosome and/or transport of the composition of the invention, or itscomponents, from the endosome to the cytoplasm of the cell. Exemplaryendosomolytic ligands include the GALA peptide (Subbarao et al.,Biochemistry, 1987, 26: 2964-2972, which is incorporated by reference inits entirety), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996,118: 1581-1586, which is incorporated by reference in its entirety), andtheir derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, which is incorporated by reference in its entirety). In oneembodiment, the endosomolytic component may contain a chemical group(e.g., an amino acid) which will undergo a change in charge orprotonation in response to a change in pH. The endosomolytic componentmay be linear or branched.

Ligands can improve transport, hybridization, and specificity propertiesand may also improve nuclease resistance of the resultant natural ormodified oligoribonucleotide, or a polymeric molecule comprising anycombination of monomers described herein and/or natural or modifiedribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyamino acids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases or a chelator(e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the oligonucleotide into the cellby activating an inflammatory response, for example. Exemplary ligandsthat would have such an effect include tumor necrosis factor alpha(TNF-alpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, naproxen or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. In another alternative, the peptide moiety caninclude a hydrophobic membrane translocation sequence (MTS). Anexemplary hydrophobic MTS-containing peptide is RFGF having the aminoacid sequence AAVALLPAVLLALLAP (SEQ ID NO: 1). An RFGF analogue (e.g.,amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) containing a hydrophobicMTS can also be a targeting moiety. The peptide moiety can be a“delivery” peptide, which can carry large polar molecules includingpeptides, oligonucleotides, and protein across cell membranes. Forexample, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ IDNO: 4)) have been found to be capable of functioning as deliverypeptides. A peptide or peptidomimetic can be encoded by a randomsequence of DNA, such as a peptide identified from a phage-displaylibrary, or one-bead-one-compound (OBOC) combinatorial library (Lam etal., Nature, 354:82-84, 1991, which is incorporated by reference in itsentirety). Preferably the peptide or peptidomimetic tethered to an iRNAagent via an incorporated monomer unit is a cell targeting peptide suchas an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized. An RGDpeptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002, which is incorporated by reference in itsentirety). An RGD peptide can facilitate targeting of an iRNA agent totumors of a variety of other tissues, including the lung, kidney,spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001,which is incorporated by reference in its entirety). Preferably, the RGDpeptide will facilitate targeting of an iRNA agent to the kidney. TheRGD peptide can be linear or cyclic, and can be modified, e.g.,glycosylated or methylated to facilitate targeting to specific tissues.For example, a glycosylated RGD peptide can deliver an iRNA agent to atumor cell expressing avB3 (Haubner et al., Jour. Nucl. Med.,42:326-336, 2001, which is incorporated by reference in its entirety).Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogenesis. Preferred conjugates of this type ligands that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003, which is incorporated by reference in its entirety).

In one embodiment, a targeting peptide can be an amphipathic α-helicalpeptide. Exemplary amphipathic α-helical peptides include, but are notlimited to, cecropins, lycotoxins, paradaxins, buforin, CPF,bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clavapeptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an aptamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polycations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligand conjugates amenable to the invention are described in U.S.patent application Ser. No. 10/916,185, filed Aug. 10, 2004; Ser. No.10/946,873, filed Sep. 21, 2004; Ser. No. 10/833,934, filed Aug. 3,2007; Ser. No. 11/115,989 filed Apr. 27, 2005 and Ser. No. 11/944,227filed Nov. 21, 2007, which are incorporated by reference in theirentireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether, e.g. a carrier described herein. The ligand ortethered ligand may be present on a monomer when said monomer isincorporated into the growing strand. In some embodiments, the ligandmay be incorporated via coupling to a “precursor” monomer after said“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporatedinto a growing oligonucleotide strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor monomer bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor monomer's tether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction may be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

Any suitable ligand in the field of RNA interference may be used,although the ligand is typically a carbohydrate e.g. monosaccharide(such as GalNAc), disaccharide, trisaccharide, tetrasaccharide,polysaccharide.

Linkers that conjugate the ligand to the nucleic acid include thosediscussed above. For example, the ligand can be one or more GalNAc(N-acetylglucosamine) derivatives attached through a monovalent,bivalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to abivalent and trivalent branched linkers include the structures shown inany of formula (IV)-(VII):

wherein:

q^(2A), q_(2B), q^(3A), q^(3B), q^(4A), q^(4B), q^(5A), q^(5B) andq^(5C) represent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;

p^(2A), p^(2B), p^(3A), p^(3B), p^(4A), p^(4B), p^(5A), p^(5B), p^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(5A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and

R^(a) is H or amino acid side chain.

Trivalent conjugating GalNAc derivatives are particularly useful for usewith RNAi agents for inhibiting the expression of a target gene, such asthose of formula (VII):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thefollowing compounds:

Definitions

As used herein, the terms “dsRNA”, “siRNA”, and “iRNA agent” are usedinterchangeably to agents that can mediate silencing of a target RNA,e.g., mRNA, e.g., a transcript of a gene that encodes a protein. Forconvenience, such mRNA is also referred to herein as mRNA to besilenced. Such a gene is also referred to as a target gene. In general,the RNA to be silenced is an endogenous gene or a pathogen gene. Inaddition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also betargeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an siRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed. Thenon-target sequences typically differ by at least 5 nucleotides.

In one embodiment, a dsRNA agent of the invention is “sufficientlycomplementary” to a target RNA, e.g., a target mRNA, such that the dsRNAagent silences production of protein encoded by the target mRNA. Inanother embodiment, the dsRNA agent of the invention is “exactlycomplementary” to a target RNA, e.g., the target RNA and the dsRNAduplex agent anneal, for example to form a hybrid made exclusively ofWatson-Crick base pairs in the region of exact complementarity. A“sufficiently complementary” target RNA can include an internal region(e.g., of at least 10 nucleotides) that is exactly complementary to atarget RNA. Moreover, in some embodiments, the dsRNA agent of theinvention specifically discriminates a single-nucleotide difference. Inthis case, the dsRNA agent only mediates RNAi if exact complementary isfound in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) for example of length less than 100, 200, 300, or400 nucleotides.

The term ‘BNA’ refers to bridged nucleic acid, and is often referred asconstrained or inaccessible RNA. BNA can contain a 5-, 6-membered, oreven a 7-membered bridged structure with a “fixed” C3′-endo sugarpuckering. The bridge is typically incorporated at the 2′-, 4′-positionof the ribose to afford a 2′, 4′-BNA nucleotide (e.g., LNA, or ENA).Examples of BNA nucleotides include the following nucleosides:

The term INA′ refers to locked nucleic acid, and is often referred asconstrained or inaccessible RNA. LNA is a modified RNA nucleotide. Theribose moiety of an LNA nucleotide is modified with an extra bridge(e.g., a methylene bridge or an ethylene bridge) connecting the 2′hydroxyl to the 4′ carbon of the same ribose sugar. For instance, thebridge can “lock” the ribose in the 3′-endo North) conformation:

The term ‘ENA’ refers to ethylene-bridged nucleic acid, and is oftenreferred as constrained or inaccessible RNA.

The “cleavage site” herein means the backbone linkage in the target geneor the sense strand that is cleaved by the RISC mechanism by utilizingthe iRNA agent. And the target cleavage site region comprises at leastone or at least two nucleotides on both side of the cleavage site. Forthe sense strand, the cleavage site is the backbone linkage in the sensestrand that would get cleaved if the sense strand itself was the targetto be cleaved by the RNAi mechanism. The cleavage site can be determinedusing methods known in the art, for example the 5′-RACE assay asdetailed in Soutschek et al., Nature (2004) 432, 173-178, which isincorporated by reference in its entirety. As is well understood in theart, the cleavage site region for a conical double stranded RNAi agentcomprising two 21-nucleotides long strands (wherein the strands form adouble stranded region of 19 consecutive base pairs having 2-nucleotidesingle stranded overhangs at the 3′-ends), the cleavage site regioncorresponds to positions 9-12 from the 5′-end of the sense strand.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to saturated and unsaturatednon-aromatic hydrocarbon chains that may be a straight chain or branchedchain, containing the indicated number of carbon atoms (these includewithout limitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₁₀ indicates that the groupmay have from 1 to 10 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an amino. The term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S— alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include trizolyl, tetrazolyl, piperazinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkyl sulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent can be further substituted.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodimentof the dsRNA agent according to the present invention, the cleavablelinking group is cleaved at least 10 times or more, preferably at least100 times faster in the target cell or under a first reference condition(which can, e.g., be selected to mimic or represent intracellularconditions) than in the blood of a subject, or under a second referencecondition (which can, e.g., be selected to mimic or represent conditionsfound in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups is redox cleavable linking groups,which may be used in the dsRNA agent according to the present invention.that are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups, which may be used in the dsRNAagent according to the present invention, are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups, which may be used in the dsRNA agentaccording to the present invention, are linking groups that are cleavedunder acidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups, which may be used in the dsRNAagent according to the present invention, are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups, which may be used in the dsRNAagent according to the present invention, are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkenylene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above. As used herein,“carbohydrate” refers to a compound which is either a carbohydrate perse made up of one or more monosaccharide units having at least 6 carbonatoms (which may be linear, branched or cyclic) with an oxygen, nitrogenor sulfur atom bonded to each carbon atom; or a compound having as apart thereof a carbohydrate moiety made up of one or more monosaccharideunits each having at least six carbon atoms (which may be linear,branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded toeach carbon atom. Representative carbohydrates include the sugars(mono-, di-, tri- and oligosaccharides containing from about 4-9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C₅and above (preferably C₅-C₈) sugars; di- and trisaccharides includesugars having two or three monosaccharide units (preferably C₅-C₈).

The present invention further relates to a use of a dsRNA agent asdefined herein for inhibiting expression of a target gene. In oneembodiment, the present invention further relates to a use of a dsRNAagent for inhibiting expression of a target gene in vitro.

The present invention further relates to a dsRNA agent as defined hereinfor use in inhibiting expression of a target gene in a subject. Thesubject may be any animal, such as a mammal, e.g., a mouse, a rat, asheep, a cattle, a dog, a cat, or a human.

In one embodiment, the dsRNA agent of the invention is administered inbuffer.

In one embodiment, siRNA compounds described herein can be formulatedfor administration to a subject. A formulated siRNA composition canassume a variety of states. In some examples, the composition is atleast partially crystalline, uniformly crystalline, and/or anhydrous(e.g., less than 80, 50, 30, 20, or 10% water). In another example, thesiRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the siRNAcomposition is formulated in a manner that is compatible with theintended method of administration, as described herein. For example, inparticular embodiments the composition is prepared by at least one ofthe following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A siRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a siRNA,e.g., a protein that complexes with siRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the siRNA preparation includes another siRNAcompound, e.g., a second siRNA that can mediate RNAi with respect to asecond gene, or with respect to the same gene. Still other preparationcan include at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent siRNA species. Such siRNAs can mediate RNAi with respect to asimilar number of different genes.

In one embodiment, the siRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than a RNA or a DNA). Forexample, a siRNA composition for the treatment of a viral disease, e.g.,HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a siRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Exemplary formulations which can be used for administering the dsRNAagent according to the present invention are discussed below.

Liposomes.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNAs, and such practice is within the invention. AnsiRNA compound, e.g., a double-stranded siRNA compound, or ssiRNAcompound, (e.g., a precursor, e.g., a larger siRNA compound which can beprocessed into a ssiRNA compound, or a DNA which encodes an siRNAcompound, e.g., a double-stranded siRNA compound, or ssiRNA compound, orprecursor thereof) preparation can be formulated for delivery in amembranous molecular assembly, e.g., a liposome or a micelle. As usedherein, the term “liposome” refers to a vesicle composed of amphiphiliclipids arranged in at least one bilayer, e.g., one bilayer or aplurality of bilayers. Liposomes include unilamellar and multilamellarvesicles that have a membrane formed from a lipophilic material and anaqueous interior. The aqueous portion contains the siRNA composition.The lipophilic material isolates the aqueous interior from an aqueousexterior, which typically does not include the siRNA composition,although in some examples, it may. Liposomes are useful for the transferand delivery of active ingredients to the site of action. Because theliposomal membrane is structurally similar to biological membranes, whenliposomes are applied to a tissue, the liposomal bilayer fuses withbilayer of the cellular membranes. As the merging of the liposome andcell progresses, the internal aqueous contents that include the siRNAare delivered into the cell where the siRNA can specifically bind to atarget RNA and can mediate RNAi. In some cases the liposomes are alsospecifically targeted, e.g., to direct the siRNA to particular celltypes.

A liposome containing a siRNA can be prepared by a variety of methods.In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the siRNA andcondense around the siRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of siRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Felgner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355;5,171,678; Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984, which are incorporated by reference in theirentirety. Commonly used techniques for preparing lipid aggregates ofappropriate size for use as delivery vehicles include sonication andfreeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys.Acta 858:161, 1986, which is incorporated by reference in its entirety).Microfluidization can be used when consistently small (50 to 200 nm) andrelatively uniform aggregates are desired (Mayhew, et al. Biochim.Biophys. Acta 775:169, 1984, which is incorporated by reference in itsentirety). These methods are readily adapted to packaging siRNApreparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274, which is incorporated byreference in its entirety).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andinclude U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640;WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl.Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon,Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver siRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated siRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of siRNA (see, e.g.,Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA,which are incorporated by reference in their entirety).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991, which is incorporated by reference in its entirety). Forcertain cell lines, these liposomes containing conjugated cationiclipids, are said to exhibit lower toxicity and provide more efficienttransfection than the DOTMA-containing compositions. Other commerciallyavailable cationic lipid products include DMRIE and DMRIE-HP (Vical, LaJolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc.,Gaithersburg, Md.). Other cationic lipids suitable for the delivery ofoligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer siRNA, into the skin. In some implementations,liposomes are used for delivering siRNA to epidermal cells and also toenhance the penetration of siRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino,R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T.et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176,1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527,1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA84:7851-7855, 1987, which are incorporated by reference in theirentirety).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith siRNA are useful for treating a dermatological disorder.

Liposomes that include siRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transfersomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include siRNA can bedelivered, for example, subcutaneously by infection in order to deliversiRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransfersomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Surfactants. For ease of exposition the formulations, compositions andmethods in this section are discussed largely with regard to unmodifiedsiRNA compounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., modified siRNA compounds, and such practice is within the scope ofthe invention. Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes (see above). siRNA(or a precursor, e.g., a larger dsiRNA which can be processed into asiRNA, or a DNA which encodes a siRNA or precursor) compositions caninclude a surfactant. In one embodiment, the siRNA is formulated as anemulsion that includes a surfactant. The most common way of classifyingand ranking the properties of the many different types of surfactants,both natural and synthetic, is by the use of the hydrophile/lipophilebalance (HLB). The nature of the hydrophilic group provides the mostuseful means for categorizing the different surfactants used informulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and other Membranous Formulations.

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified siRNA compounds. It may be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other siRNA compounds, e.g., modified siRNA compounds,and such practice is within the invention. The siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof)) composition can be provided as a micellar formulation.“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Particles.

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to modifiedsiRNA compounds. It may be understood, however, that these particles,formulations, compositions and methods can be practiced with other siRNAcompounds, e.g., unmodified siRNA compounds, and such practice is withinthe invention. In another embodiment, an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor,e.g., a larger siRNA compound which can be processed into a ssiRNAcompound, or a DNA which encodes an siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, or precursorthereof) preparations may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

Pharmaceutical Compositions

The iRNA agents of the invention may be formulated for pharmaceuticaluse. The present invention further relates to a pharmaceuticalcomposition comprising the dsRNA agent as defined herein.Pharmaceutically acceptable compositions comprise atherapeutically-effective amount of one or more of the dsRNA agents inany of the preceding embodiments, taken alone or formulated togetherwith one or more pharmaceutically acceptable carriers (additives),excipient and/or diluents.

The pharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally. Delivery using subcutaneous or intravenous methods can beparticularly advantageous.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe invention which is effective for producing some desired therapeuticeffect in at least a sub-population of cells in an animal at areasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium state, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; and (22) other non-toxic compatible substancesemployed in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 0.1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aiRNA, e.g., a protein that complexes with iRNA to form an iRNP. Stillother agents include chelators, e.g., EDTA (e.g., to remove divalentcations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broadspecificity RNAse inhibitor such as RNAsin) and so forth.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure. The patient receiving this treatment is any animal in need,including primates, in particular humans, and other mammals such asequines, cattle, swine and sheep; and poultry and pets in general.

Double-stranded RNAi agents are produced in a cell in vivo, e.g., fromexogenous DNA templates that are delivered into the cell. For example,the DNA templates can be inserted into vectors and used as gene therapyvectors. Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection, local administration (U.S. Pat. No.5,328,470, which is incorporated by reference in its entirety), or bystereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057, which is incorporated by reference in itsentirety). The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. The DNA templates, for example, can include two transcriptionunits, one that produces a transcript that includes the top strand of adsRNA agent and one that produces a transcript that includes the bottomstrand of a dsRNA agent. When the templates are transcribed, the dsRNAagent is produced, and processed into siRNA agent fragments that mediategene silencing.

Routes of Delivery

The dsRNA agent as defined herein or a pharmaceutical compositioncomprising a dsRNA agent as defined herein can be administered to asubject using different routes of delivery. A composition that includesan iRNA can be delivered to a subject by a variety of routes. Exemplaryroutes include: intravenous, subcutaneous, topical, rectal, anal,vaginal, nasal, pulmonary, ocular.

The iRNA molecules and/or the dsRNA agent of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically include one or more speciesof iRNA and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The compositions of the present invention may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. Administration may be topical(including ophthalmic, vaginal, rectal, intranasal, transdermal), oralor parenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection, or intrathecalor intraventricular administration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Dosage

In one aspect, the invention features a method of administering a dsRNAagent, e.g., a siRNA agent, to a subject (e.g., a human subject). Inanother aspect, the present invention relates to a dsRNA agent asdefined herein for use in inhibiting expression of a target gene in asubject. The method or the medical use includes administering a unitdose of the dsRNA agent, e.g., a siRNA agent, e.g., double strandedsiRNA agent that (a) the double-stranded part is 14-40 nucleotides (nt)long, for example, 21-23 nt, (b) is complementary to a target RNA (e.g.,an endogenous or pathogen target RNA), and, optionally, (c) includes atleast one 3′ overhang 1-5 nucleotide long. In one embodiment, the unitdose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1,0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g.,about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750,300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015,0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, ora topical application. In some embodiments dosages may be less than 10,5, 2, 1, or 0.1 mg/kg of body weight.

In some embodiments, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than adsRNA agent, e.g., other than a siRNA agent. In another embodiment, thesubject has atherosclerosis and the effective dose of a dsRNA agent,e.g., a siRNA agent, is administered in combination with, e.g., aftersurgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a dsRNA agent, e.g., a siRNA agent, (e.g., aprecursor, e.g., a larger dsRNA agent which can be processed into asiRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent,or precursor thereof). The maintenance dose or doses can be the same orlower than the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10,1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. Themaintenance doses are, for example, administered no more than once every2, 5, 10, or 30 days. Further, the treatment regimen may last for aperiod of time which will vary depending upon the nature of theparticular disease, its severity and the overall condition of thepatient. In certain embodiments the dosage may be delivered no more thanonce per day, e.g., no more than once per 24, 36, 48, or more hours,e.g., no more than once for every 5 or 8 days. Following treatment, thepatient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the composition includes a plurality of dsRNA agentspecies. In another embodiment, the dsRNA agent species has sequencesthat are non-overlapping and non-adjacent to another species withrespect to a naturally occurring target sequence. In another embodiment,the plurality of dsRNA agent species is specific for different naturallyoccurring target genes. In another embodiment, the dsRNA agent is allelespecific.

The dsRNA agents of the invention described herein can be administeredto mammals, particularly large mammals such as nonhuman primates orhumans in a number of ways.

In one embodiment, the administration of the dsRNA agent, e.g., a siRNAagent, composition is parenteral, e.g., intravenous (e.g., as a bolus oras a diffusible infusion), intradermal, intraperitoneal, intramuscular,intrathecal, intraventricular, intracranial, subcutaneous, transmucosal,buccal, sublingual, endoscopic, rectal, oral, vaginal, topical,pulmonary, intranasal, urethral or ocular. Administration can beprovided by the subject or by another person, e.g., a health careprovider. The medication can be provided in measured doses or in adispenser which delivers a metered dose. Selected modes of delivery arediscussed in more detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of dsRNA agents described herein

In particular embodiments, the present invention relates to the dsRNAagents of the present invention for use in the methods described above.

Methods of Inhibiting Expression of the Target Gene

Embodiments of the invention also relate to methods for inhibiting theexpression of a target gene. The method comprises the step ofadministering the dsRNA agents in any of the preceding embodiments, inan amount sufficient to inhibit expression of the target gene. Thepresent invention further relates to a use of a dsRNA agent as definedherein for inhibiting expression of a target gene in a target cell. In apreferred embodiment, the present invention further relates to a use ofa dsRNA agent for inhibiting expression of a target gene in a targetcell in vitro.

Another aspect the invention relates to a method of modulating theexpression of a target gene in a cell, comprising providing to said cella dsRNA agent of this invention. In one embodiment, the target gene isselected from the group consisting of Factor VII, Eg5, PCSK9, TPX2,apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene,GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene,PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin,Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene,Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKBgene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene,topoisomerase II alpha gene, mutations in the p73 gene, mutations in thep21(WAF1/CIP1) gene, mutations in the p27(KIP1) gene, mutations in thePPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene,mutations in the MIB I gene, mutations in the MTAI gene, mutations inthe M68 gene, mutations in tumor suppressor genes, and mutations in thep53 tumor suppressor gene.

In particular embodiments, the present invention relates to the dsRNAagents of the present invention for use in the methods described above.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1. In Vitro Screening of siRNA Duplexes

Cell Culture and Transfections:

Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grownto near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNAmixture. Cells were incubated for either 24 or 120 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1 nMfinal duplex concentration and dose response experiments were done using8, 4 fold serial dilutions with a maximum dose of 10 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixingspeed was the same throughout the process). Ten microliters of magneticbeads and 80 μl Lysis/Binding Buffer mixture were added to a roundbottom plate and mixed for 1 minute. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, the lysed cells were added to theremaining beads and mixed for 5 minutes. After removing supernatant,magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixedfor 1 minute. Beads were capture again and supernatant removed. Beadswere then washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂Oper reaction were added into 5 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)),0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_m1 (human)cat #Rn00562124_m1 (rat)) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well plate (Roche cat#04887301001). Real time PCR was done in a Roche LC 480 Real Time PCRmachine (Roche). Each duplex was tested in at least two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with AD-1955 or naïve cells over the same dose range,or to its own lowest dose. IC₅₀s were calculated for each individualtransfection as well as in combination, where a single IC₅₀ was fit tothe data from both transfections.

The results of gene silencing of the exemplary siRNA duplex with variousmotif modifications of the invention are shown in the table below.

Example 2. RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis:

All oligonucleotides were synthesized on an AKTAoligopilot synthesizeror an ABI 394 synthesizer. Commercially available controlled pore glasssolid support (dT-CPG, 500A, Prime Synthesis) and RNA phosphoramiditeswith standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to trans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. Fluorophore labeledsiRNAs were synthesized from the corresponding phosphoramidite or solidsupport, purchased from Biosearch Technologies. The oleyl lithocholic(GalNAc)₃ polymer support made in house at a loading of 38.6 μmol/gram.The Mannose (Man)₃ polymer support was also made in house at a loadingof 42.0 μmol/gram.

Conjugation of the ligand of choice at desired position, for example atthe 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 minutes coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 minutes oxidationwait time conjugated oligonucleotide. Phosphorothioate was introduced bythe oxidation of phosphite to phosphorothioate by using a sulfurtransfer reagent such as DDTT (purchased from AM Chemicals), PADS and orBeaucage reagent The cholesterol phosphoramidite was synthesized inhouse, and used at a concentration of 0.1 M in dichloromethane. Couplingtime for the cholesterol phosphoramidite was 16 minutes.

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. siRNA Preparation

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 minutes and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLCanalysis.

Example 3: In Vitro Silencing Activity with Various ChemicalModifications on ANGPTL3 siRNA

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS,streptomycin, and glutamine (ATCC) before being released from the plateby trypsinization. Transfection was carried out by adding 14.8 μl ofOpti-MEM plus 0.41 of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well intoa 96-well plate and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2×10⁴ Hep3Bcells were then added to the siRNA mixture. Cells were incubated foreither 24 or 120 hours prior to RNA purification. Single doseexperiments were performed at 10 nM and 0.1 nM final duplexconcentration and dose response experiments were done at 10, 1, 0.5,0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nMfinal duplex concentration unless otherwise stated.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 41 Random primers, 1μl Reverse

Transcriptase, 1 μl RNase inhibitor and 3.41 of H₂O per reaction wereadded into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 orS-1000 thermal cycler (Hercules, Calif.) through the following steps:25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real time PCR

2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqManProbe

(Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (AppliedBiosystems cat #Hs00205581 ml) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat#04887301001). Real time PCR was done in an ABI 7900HT Real Time PCRsystem (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex wastested in two independent transfections, and each transfection wasassayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data was analyzed using theΔΔCt method and normalized to assays performed with cells transfectedwith 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculatedusing a 4 parameter fit model using XLFit and normalized to cellstransfected with AD-1955 or naïve cells over the same dose range, or toits own lowest dose. AD-1955 sequence, used as a negative control,targets luciferase and has the following sequence:

sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 5);

antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 6).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

Example 4: Chemical Modifications on siRNAs and In Vitro Silencing ofthe Modified siRNAs

Sense Strand Design

Ligand Design and Site of Conjugation

The sense strand was conjugated to the GalNAc ligand at the 3′-position,same as the parent compound.

Sense Position 11

Sense strand position 11 at the putative cleavage site (opposite ASposition 11, when the sense strand is 21 nucleotides in length, and theantisense strand is 23 nucleotides in length) was modified with anuclease sensitive modification (e.g. DNA). Data from statisticalanalysis across many different conjugates suggests importance of thisposition.

Thermal Destabilization of Sense 3′-Region (Positions 16-18):

This region was modified with thermally destabilizing modifications,such as GNA or mismatch to opposite AS-strand. Modifications of position16 or 17 appeared to be most impactful. FIG. 1 and Table 1 highlightthis position/region and the effect of thermal destabilization on invitro efficacy. Efficacious knockdown comparable to parent templatedesign was obtained with GNA or other thermally destabilizingmodifications, such as abasic (Y34) or mismatches to the antisensestrand. On the other hand, reduced silencing was generally observed for2′-OMe or DNA modifications complementary to the opposite AS-strand.

TABLE 1 Sense strand position 17: Sequence and modificationsof the siRNAs evaluated in vitro (see FIG. 1).Table 1 discloses SEQ ID NOS 7-74, respectively, in order of columns.Tar- Duplex Sense Antisense Modifi- 10 0.1 get ID Sense ID (5′ to 3′)AS ID (5′ to 3′) cation nM SD nM SD mTTR AD- A- AfsasCfaGfuGf A-usUfsaUfaGfa parent  3.4  1.2  12.6  3.6 57727.21 117799.49uUfCfUfuGfcUf 117800.20 GfcAfagaAfcA cUfaUfaAfL96 fcUfgUfususu mTTR AD-A- asascaguguucd A- usUfsaUfaGfa 2′-OMe 46.3  6.8  39.3 13.7 61291.1123259.1 Tugcucuauaa 117800.29 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usUfsaUfaGfa DNA 43.0 10.0  35.2  8.9 61297.1 123260.1TugcucdTauaa 117800.30 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usUfsaUfaGfa GNA 14.9  6.4  19.1  7.0 61377.1 123316.1Tugcuc(Tgn)au 117800.38 GfcAfagaAfcA aaL96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usdTsauagagc DNA 27.1  9.6  43.5 19.9 61364.1 123260.9TugcucdTauaa 123268.5 dAagadAcacug L96 uususu mTTR AD- A- asascaguguucdA- usdTsauagagc GNA 13.4  6.2  17.8  4.8 61398.1 123316.9 Tugcuc(Tgn)au123268.6 dAagadAcacug aaL96 uususu mTTR AD- A- AfsasCfaGfuGf A-usUfsaUfaGfa parent 10.3  3.1  43.0  5.6 57727.41 117799.131uUfCfUfuGfcUf 117800.71 GfcAfagaAfcA cUfaUfaAfL96 fcUfgUfususu mTTR AD-A- asascaguguucd A- usUfsaUfaGfa abasic 18.1  2.7  51.5 10.0 64426.1128287.1 TugcucY34auaa 117800.76 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD-A- asascaguguucd A- usUfsaUfaGfa a:a mm 33.8  3.4  50.9  4.9 64392.1128288.1 Tugcucaauaa 117800.77 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usUfsaUfaGfa c:a mm 27.3  4.2  49.4  8.1 64397.1128289.1 Tugcuccauaa 117800.78 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usUfsaUfaGfa g:a mm 34.6  3.7  62.0  8.4 64403.1128290.1 Tugcucgauaa 117800.79 GfcAfagaAfcA L96 fcUfgUfususu mTTR AD- A-asascaguguucd A- usdTsauagagc RNA 56.8  7.4  90.2 15.6 64411.1 128305.1TugcucUauaa 123268.13 dAagadAcacug L96 uususu mTTR AD- A- asascaguguucdA- usdTsauagagc g:a mm 14.7  1.9  64.3  3.5 64421.1 128290.2 Tugcucgauaa123268.15 dAagadAcacug L96 uususu ANG AD- A- AfscsAfuAfuUf A-asAfsaAfaGfa parent  7.5  1.9  29.6  7.0 57927.7 117426.25 uGfAfUfcAfgUf117427.23 CfuGfaucAfaA cUfuUfuUfL96 fuAfuGfususg ANG AD- A-ascsauauuugad A- asAfsaAfaGfa GNA at 11.9  7.7  37.5 12.1 63144.2126526.4 Tcaguc(Tgn)uu 117427.28 CfuGfaucAfaA 17 uuL96 fuAfuGfususg ANGAD- A- ascsauauuugad A- asAfsaAfaGfa DNA 16.6  1.4  57.4  9.8 64744.1129588.1 TcagucdTuuuu 117427.31 CfuGfaucAfaA L96 fuAfuGfususg ANG AD- A-ascsauauuugad A- asAfsaAfaGfa dG:A mm 12.7  5.7  36.7  6.4 64761.1129591.1 TcagucdGuuuu 117427.34 CfuGfaucAfaA L96 fuAfuGfususg ANG AD- A-ascsauauuugad A- asAfsaAfaGfa dC:A mm 13.2  5.8  39.0  9.5 64767.1129592.1 TcagucdCuuuu 117427.35 CfuGfaucAfaA L96 fuAfuGfususg ANG AD- A-ascsauauuugad A- asAfsaAfaGfa dA:A mm 11.8  2.7  50.0  1.7 64772.1129593.1 TcagucdAuuuu 117427.36 CfuGfaucAfaA L96 fuAfuGfususg ApoC3 AD-A- GfscsUfuAfaAf A- asGfsaAfuAfc parent  8.2  3.8  45.0 24.0 64787.1117361.23 aGfGfGfaCfaGf 129546.17 UfgUfcccUfuU uAfuUfcUfL96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc 2′-OMe 33.1  7.7 110.5  9.364823.1 129551.2 Gacaguauucu 129546.21 UfgUfcccUfuU L96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc DNA 52.1 13.8 103.8 28.764806.1 129556.2 GacagudAuucu 129546.26 UfgUfcccUfuU L96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc GNA  8.7  2.1  50.2 15.964794.1 129554.4 Gacagu(Agn)uu 129546.24 UfgUfcccUfuU cuL96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc g:u mm 25.5 10.3  75.8 18.464829.1 129560.2 Gacaguguucu 129546.30 UfgUfcccUfuU L96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc c:u mm 21.8  6.1  39.7  5.764789.1 129561.2 Gacagucuucu 129546.31 UfgUfcccUfuU L96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc u:u mm 19.9  6.3  54.2 10.164795.1 129562.2 Gacaguuuucu 129546.32 UfgUfcccUfuU L96 fuAfaGfcsasaApoC3 AD- A- gscsuuaaaaggd A- asGfsaAfuAfc dT:u mm 18.2  6.1  59.3 19.964812.1 129557.4 GacagudTuucu 129546.27 UfgUfcccUfuU L96 fuAfaGfcsasaTTRSC AD- A- UfsgsGfgAfuUf A- usCfsuUfgGfu parent 17.3 10.2  72.2 13.564474.1 128493.1 uCfAfUfgUfaAf 128494.1 UfaCfaugAfaA cCfaAfgAfL96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu 2′-OMe 43.3 25.6100.1  9.6 64493.1 128505.1 Tguaaccaaga 128494.8 UfaCfaugAfaA L96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu DNA 18.7  6.2 81.0  8.7 64460.1 128507.1 TguaacdCaaga 128494.10 UfaCfaugAfaA L96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu GNA 26.1 12.9 68.0 14.8 64455.1 128506.1 Tguaac(Cgn)aa 128494.9 UfaCfaugAfaA gaL96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu u:g mm 19.0  8.2 96.2 14.5 64482.1 128511.1 Tguaacuaaga 128494.14 UfaCfaugAfaA L96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu a:g mm 19.4  4.1 72.6  7.9 64488.1 128512.1 Tguaacaaaga 128494.15 UfaCfaugAfaA L96fuCfcCfasusc TTRSC AD- A- usgsggauuucad A- usCfsuUfgGfu g:g mm 29.8 11.6 93.9 13.1 64494.1 128513.1 Tguaacgaaga 128494.16 UfaCfaugAfaA L96fuCfcCfasusc

FIG. 2 and Table 2 showed the positional effect of the thermallydestabilizing modification GNA across the 3′-region (positions 16-18).The results indicate that GNA modifications in positions 16 and 17showed good efficacy, similar to the parent design, while GNA inposition 18 showed a decrease in activity.

TABLE 2 Positional effect of thermal destabilization with GNA:Sequence and modifications of the siRNAs evaluated invitro (see FIG. 2). Table 2 discloses SEQ ID NOS 75-90,respectively, in order of columns. Tar- Duplex Sense Antisense Modifi-10 0.1 get ID Sense ID (5′ to 3′) AS ID (5′ to 3′) cation nM SD nM SDANG AD- A- AfscsAfuAf A- asAfsaAfaGfaCfuGfa parent  7.5  1.9 29.6  7.0579277 117426.25 uUfuGfAfUf 117427.23 ucAfaAfuAfuGfususg cAfgUfcUfuUfuUfL96 ANG AD- A- ascsauauuu A- asAfsaAfaGfaCfuGfa GNA at 11.3  5.438.8 11.3 64766.1 129585.1 gadTcagu 117427.27 ucAfaAfuAfuGfususg 16(Cgn)uuuuu L96 ANG AD- A- ascsauauuu A- asAfsaAfaGfaCfuGfa GNA at 11.9 7.7 37.5 12.1 63144.2 126526.4 gadTcaguc 117427.28 ucAfaAfuAfuGfususg17 (Tgn)uuuu L96 ANG AD- A- ascsauauuu A- asAfsaAfaGfaCfuGfa GNA at 19.8 3.7 65.8  6.8 64777.1 129586.1 gadTcagucu 117427.29 ucAfaAfuAfuGfususg18 (Tgn)uuu L96 ApoC3 AD- A- GfscsUfuAf A- asGfsaAfuAfcUfgUfc parent 8.2  3.8 45.0 24.0 64787.1 117361.23 aAfaGfGfGf 129546.17ccUfuUfuAfaGfcsasa aCfaGfuAfu UfcUfL96 ApoC3 AD- A- gscsuuaaaa A-asGfsaAfuAfcUfgUfc GNA at 15.9  3.4 69.2 11.6 64788.1 129553.5 ggdGacag129546.23 ccUfuUfuAfaGfcsasa 16 (Tgn)auucu L96 ApoC3 AD- A- gscsuuaaaaA- asGfsaAfuAfcUfgUfc GNA at  8.7  2.1 50.2 15.9 64794.1 129554.4ggdGacagu 129546.24 ccUfuUfuAfaGfcsasa 17 (Agn)uucu L96 ApoC3 AD- A-gscsuuaaaa A- asGfsaAfuAfcUfgUfc GNA at 37.9 10.2 96.0 13.8 64800.1129555.2 ggdGacagua 129546.25 ccUfuUfuAfaGfcsasa 18 (Tgn)ucu L96

TABLE 3 Antisense position 2: Sequence and modificationsof the siRNAs evaluated in vitro (see FIG. 3). Table 3discloses SEQ ID NOS 91-118, respectively, in order of columns. DuplexSense Antisense Modifi- Target ID Sense ID (5′ to 3′) AS ID (5′ to 3′)cation 10 nM SD 0.1 nM SD mTTR AD- A- AfsasCfaGfuGf A- usUfsaUfaGfaparent  10.3  3.1  43.0  5.6 57727.41 117799.131 uUfCfUfuGfcUf 117800.71GfcAfagaAfcA cUfaUfaAfL96 fcUfgUfususu mTTR AD- A- asascaguguucd A-usdTsauagagc DNA  15.3  2.2  49.6  6.5 61398.5 123316.21 Tugcuc(Tgn)au123268.11 dAagadAcacug aaL96 uususu mTTR AD- A- asascaguguucd A-ususauagagcd 2′-OMe  42.4  5.0  79.7  6.8 64393.1 123316.23Tugcuc(Tgn)au 128294.1 AagadAcacugu aaL96 ususu mTTR AD- A-asascaguguucd A- usUsauagagcd RNA  19.1  1.6  61.7  9.9 64415.1123316.27 Tugcuc(Tgn)au 128298.1 AagadAcacugu aaL96 ususu ApoC3 AD- A-GfscsUfuAfaAf A- asGfsaAfuAfc Parent   8.2  3.8  45.0 24.0 64787.1117361.23 aGfGfGfaCfaGf 129546.17 UfgUfcccUfuU uAfuUfcUfL96 fuAfaGfcsasaApoC3 AD- A- GfscsUfuAfaAf A- asdGsaauacug DNA  34.2 11.0  63.9 10.164802.1 117361.33 aGfGfGfaCfaGf 129571.5 dTcccdTuuuaa uAfuUfcUfL96gcsasa ApoC3 AD- A- GfscsUfuAfaAf A- asgsaauacugd 2′-OMe 131.8 46.9 84.5  8.5 64790.1 117361.31 aGfGfGfaCfaGf 129569.2 TcccdTuuuaaguAfuUfcUfL96 csasa TTRSC AD- A- UfsgsGfgAfuUf A- usCfsuUfgGfu Parent 17.3 10.2  72.2 13.5 64474.1 128493.1 uCfAfUfgUfaAf 128494.1UfaCfaugAfaA cCfaAfgAfL96 fuCfcCfasusc TTRSC AD- A- UfsgsGfgAfuUf A-usdCsuugguua DNA  24.8 13.1  84.3 27.0 64472.1 128493.14 uCfAfUfgUfaAf128525.1 dCaugdAaaucc cCfaAfgAfL96 casusc TTRSC AD- A- UfsgsGfgAfuUf A-uscsuugguuad 2′-OMe  39.9 17.1 126.6 44.6 64484.1 128493.16uCfAfUfgUfaAf 128527.1 CaugdAaauccc cCfaAfgAfL96 asusc TTRSC AD- A-UfsgsGfgAfuUf A- usCsuugguuad RNA  88.6 31.5 102.3 28.1 64496.1128493.18 uCfAfUfgUfaAf 128529.1 CaugdAaauccc cCfaAfgAfL96 asusc TMP AD-A- CfsusGfgUfaUf A- usUfsgUfaCfc Parent  32.2 14.2  66.5 12.1 60940.7122745.22 uUfCfCfuAfgGf 122746.24 CfuAfggaAfaU gUfaCfaAfL96 faCfcAfgsasgTMP AD- A- csusgguauuucd A- usdTsguacccu DNA  62.4 15.4  87.2 23.864567.1 126602.4 Cuaggg(Tgn)ac 129067.1 dAggadAauacc aaL96 agsasg TMPAD- A- csusgguauuucd A- usUsguacccud RNA  48.9 16.0  88.6 37.1 64586.1126602.7 Cuaggg(Tgn)ac 129085.1 AggadAauacca aaL96 gsasgAntisense Strand Design

AS Position 2

This position has been identified by statistical analysis of largeconjugate dataset and positional walks through AS-strand as beingsensitive to sterically demanding 2′-modifications incl. 2′-OMe. Theinventors found, however, that several modifications, including DNA, insome cases RNA, as well as other modifications without steric bulk atthe 2′-position can be well tolerated in the context of non-F designs.The results from in vitro silencing studies are summarized in FIG. 3 andTable 3, indicating that DNA as well as RNA at position 2 generallymaintains activity of the non-F designs similar to the parent templatedesign, while 2′-OME is generally not well tolerated and leads toreduced activity.

AS Position 14

This position has been identified by statistical analysis of largeconjugate dataset and positional walks through AS-strand as beingsensitive to sterically demanding 2′-modifications incl. 2′-OMe. It hasbeen found, however, that several modifications, including DNA, in somecases RNA, as well as other modifications without steric bulk at the2′-position can be well tolerated in the context of non-F designs. Theresults from in vitro silencing studies are summarized in FIG. 4 andTable 4, indicating that DNA as well as RNA at position 14 generallymaintains activity of the non-F designs similar to the parent templatedesign, while 2′-OME is generally not well tolerated and leads toreduced activity.

TABLE 4 Antisense position 14: Sequence and chemistriesof siRNAs evaluated in vitro (see FIG. 4). Table 4discloses SEQ ID NOS 119-142, respectively, in order of columns. DuplexSense Antisense Modifi- Target ID Sense ID (5′ to 3′) AS ID (5′ to 3′)cation 10 nM SD 0.1 nM SD mTTR AD- A- AfsasCfaGfuGf A- usUfsaUfaGfaparent 10.3  3.1 43.0  5.6 57727.41 117799.132 uUfCfUfuGfcUf 117800.71GfcAfagaAfcA cUfaUfaAfL96 fcUfgUfususu mTTR AD- A- asascaguguucd A-usdTsauagagc DNA 15.3  2.2 49.6  6.5 61398.5 123316.21 Tugcuc(Tgn)au123268.11 dAagadAcacug aaL96 uususu mTTR AD- A- asascaguguucd A-usdTsauagagc RNA 14.3  2.2 64.2  9.0 64273.2 123316.29 Tugcuc(Tgn)au128300.1 dAagaAcacugu aaL96 ususu mTTR AD- A- asascaguguucd A-usdTsauagagc LNA 45.5  2.1 69.5  9.6 64132.1 123316.13 Tugcuc(Tgn)au128243.1 dAaga(Aln)ca aaL96 cuguususu ApoC3 AD- A- GfscsUfuAfaAf A-asGfsaAfuAfc parent  8.2  3.8 45.0 24.0 64787.1 117361.23 aGfGfGfaCfaGf129546.17 UfgUfcccUfuU uAfuUfcUfL96 fuAfaGfcsasa ApoC3 AD- A-GfscsUfuAfaAf A- asdGsaauacug DNA 34.2 11.0 63.9 10.1 64802.1 117361.33aGfGfGfaCfaGf 129571.5 dTcccdTuuuaa uAfuUfcUfL96 gcsasa ApoC3 AD- A-GfscsUfuAfaAf A- asdGsaauacug RNA 20.7  4.2 63.8  9.0 64831.1 117361.38aGfGfGfaCfaGf 129576.2 dTcccUuuuaag uAfuUfcUfL96 csasa TTRSC AD- A-UfsgsGfgAfuUf A- usCfsuUfgGfu parent 17.3 10.2 72.2 13.5 64474.1128493.1 uCfAfUfgUfaAf 128494.1 UfaCfaugAfaA cCfaAfgAfL96 fuCfcCfasuscTTRSC AD- A- UfsgsGfgAfuUf A- usdCsuugguua DNA 24.8 13.1 84.3 27.064472.1 128493.14 uCfAfUfgUfaAf 128525.1 dCaugdAaaucc cCfaAfgAfL96casusc TTRSC AD- A- UfsgsGfgAfuUf A- usdCsuugguua RNA 15.1  6.6 60.010.8 64458.1 128493.19 uCfAfUfgUfaAf 128530.1 dCaugAaauccc cCfaAfgAfL96asusc TTRSC AD- A- usgsggauuucad A- usdCsuugguua DNA 20.5 10.8 62.3 13.864515.1 128506.2 Tguaac(Cgn)aa 128525.2 dCaugdAaaucc gaL96 casusc TTRSCAD- A- usgsggauuucad A- usdCsuugguua RNA 26.0 13.2 60.2 19.1 64504.1128506.4 Tguaac(Cgn)aa 128530.2 dCaugAaauccc gaL96 asusc

TABLE 5 Sequence and chemistry of the siRNAs targetingmTTR used in the in vivo study in mice.Table 5 discloses SEQ ID NOS 143-148, respectively, in order of columns.Duplex Sense Antisense ID ID Sense (5′ to 3′) AS ID (5′ to 3′) DesignAD-57727 A-117799 AfsasCfaGfuGfuUfC A-117800 usUfsaUfaGfaGfcAfa parentfUfuGfcUfcUfaUf; gaAfcAfcUfgUfususu AD-61398 A-123316 asascaguguucdTugcA-123268 usdTsauagagcdAagad parent uc(Tgn)auaaL96 Acacuguususunon-F motif AD-64273 A-123316 asascaguguucdTugc A-128300usdTsauagagcdAagaA AS: RNA uc(Tgn)auaaL96 cacuguususu at 14In Vivo Evaluation

siRNAs Targeting mTTR

Animals (n=3/group) were administered a single siRNA dose of 2.5 mg/kgand FVII serum protein levels were measured pre-dose and at days 4, 7,13, 22, 29, and 36. FIG. 5 shows the FVII protein concentration-timeprofile for the 2 non-F siRNAs AD-61398 and 64273 in comparison to theparent compound AD-57727. In FIG. 6, the mTTR protein reduction 96 hpost dose is shown for the two non-F siRNAs at three different doselevels in comparison to the parent compounds. In FIG. 7, the profiles ofmTTR serum protein reduction is shown for repeat-dose regimen (1 mg/kg,QW) up to day 42 (total of 6 doses).

Overall, the studies indicate that the non-F siRNAs AD-61398 andAD-642733 exhibited in vivo efficacy and potency similar to the parenttemplate design.

siRNAs Targeting TMPRSS6

TABLE 6 Sequence and chemistry of siRNAs targetingTMPRSS6. Table 6 discloses SEQ ID NOS 149-158,respectively, in order of columns. Duplex Sense Antisense 10 nM 0.1 nMID Sense ID sequence AS ID sequence Avg SD Avg SD AD- A- CfsusGfgUfaUfA- usUfsgUfaCfc 32.2 14.2 66.5 12.1 60940.7 122745.22 uUfCfCfuAfgGf122746.24 CfuAfggaAfaU gUfaCfaAfL96 faCfcAfgsasg AD- A- csusgguadTuuc A-usUsguacccud 25.2 10.2 57.5 27.9 64604.1 129074.2 dCuaggg(Tgn)a 129085.2AggadAauacca caaL96 gsasg AD- A- csusggudAuuuc A- usdTsguacccu 23.0  8.674.7 17.7 64601.1 129073.1 dCuaggg(Tgn)a 129067.6 dAggadAauacc caaL96agsasg AD- A- csusgguauuucd A- usdTsguacccu 62.4 15.4 87.2 23.8 64567.1126602.4 Cuaggg(Tgn)ac 129067.1 dAggadAauacc aaL96 agsasg AD- A-csusgguadTuuc A- usdTsguacccu 49.7  9.4 49.4 19.7 64569.1 129083.1dCuagggdAacaa 129067.16 dAggadAauacc L96 agsasg

The results indicate different in vivo efficacies of the non-F designsdepending on the exact placement of modifications and combination ofsense and AS-strands. Although the in vitro data suggests that the non-Fcompounds had similar potency/efficacy to the parent, the non-F compoundAD-64604, found to be most active in vivo was still significantly lessefficacious than the parent AD-60940 (see FIG. 8).

Further refinements of the non-F design were made and evaluated assummarized in Table 7. FIG. 9 shows the TMPRSS6 mRNA silencing in liver7 days post a single SC dose of 3 mg/kg.

TABLE 7 Sequence and chemistry of 2^(nd) set of siRNAstargeting TMPRSS6. Table 7 discloses SEQ ID NOS 159-178,respectively, in order of columns. Duplex Sense Antisense ID Sense ID(5′ to 3′) AS ID (5′ to 3′) Parent AD-64601 A-129073.2 csusggudAuuucA-129067.18 usdTsguaccc AD-60940 dCuaggg(Tgn)a udAggadAaua caaL96ccagsasg AD-65105 A-129073.2 csusggudAuuuc A-129085.5 usUsguacccuAD-60940 dCuaggg(Tgn)a dAggadAauac caaL96 cagsasg AD-65106 A-129073.2csusggudAuuuc A-129086.2 usdTsguaccc AD-60940 dCuaggg(Tgn)a udAsggadAsacaaL96 uaccagsasg AD-65107 A-129710.1 csusggudAuuuc A-129067.18usdTsguaccc AD-60940 dCuagggdAacaa udAggadAaua L96 ccagsasg AD-65108A-129710.1 csusggudAuuuc A-129085.5 usUsguacccu AD-60940 dCuagggdAacaadAggadAauac L96 cagsasg AD-65109 A-129710.1 csusggudAuuuc A-129086.2usdTsguaccc AD-60940 dCuagggdAacaa udAsggadAsa L96 uaccagsasg AD-65110A-130024.1 csusggudAsuuu A-129067.18 usdTsguaccc AD-60940 dCuaggg(Tgn)audAggadAaua caaL96 ccagsasg AD-65111 A-130024.1 csusggudAsuuu A-129085.5usUsguacccu AD-60940 dCuaggg(Tgn)a dAggadAauac caaL96 cagsasg AD-65112A-130024.1 csusggudAsuuu A-129086.2 usdTsguaccc AD-60940 dCuaggg(Tgn)audAsggadAsa caaL96 uaccagsasg AD-65104 A-129875.1 usgsguadTuuccA-129876.1 usdTsuguacc AD-61002 dTagggudTcaaa cdTaggdAaau L96 accasgsa

As shown in FIG. 9, the refinement yielded at least one non-F compound(AD-65105) with in vivo efficacy comparable to the parent (AD-60940).The compound contains a sense strand with DNA at positions 6 and 11 andan anti sense strand with RNA in position 2 and DNA in positions 10, 14.

Motif Design

When designing the motif, the sense strand was conjugated to the GalNAcligand at the 3′-position, using the same procedure as used with theparent compound. Additional motifs were designed according to theembodiments of the invention. Representative sequences are listed inTable 8.

TABLE 8 Representative sequences (Table 8 discloses SEQ ID NOS 179-190,respectively, in order of appearance) Du- plex Name Strand Sequences AD-S AfsasCfaGfuGfuUfCfUfuGfcUfcUfaUfaAfL96 57727 ASusUfsaUfaGfaGfcAfagaAfcAfcUfgUfususu AD- SGfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 57553 ASasGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsAfsa AD- SCfsusUfgCfuCfuAfUfAfaAfcCfgUfgUfuAfL96 63042 ASusAfsaCfaCfgGfuUfuauAfgAfgCfaAfgsasa AD- SUfscsCfuCfuGfaUfGfGfuCfaAfaGfuCfcUfL96 63085 ASasGfsgAfcUfuUfgAfccaUfcAfgAfgGfascsa AD- SgscsuuaaAfaGfGfGfacaguauucaL96 65703 ASusGfsaauAfcUfGfucccUfuUfuaagcsasa AD- S gscsuuaaAfaGfGfGfacaguauucaL9665704 AS usGfsaauacugucccUfuuuaagcsasa

In Vitro Results

As shown in FIG. 10, across ten sequences representing three targets,two motifs, Motif 1 (six phosphorothioate internucleotide linkagemodifications to the sense and antisense strand; four 2′-F modificationsat positions 7 and 9-11 of the sense strand from 5′-end of the sensestrand, and four 2′-F modifications at positions 2, 6, 14, and 16 of theantisense strand from 5′-end of the antisense strand) and Motif 2 (sixphosphorothioate internucleotide linkage modifications to the sense andantisense strand; four 2′-F modifications at positions 7 and 9-11 of thesense strand from 5′-end of the sense strand, and six 2′-F modificationsat positions 2, 6, 8-9, 14, and 16 of the antisense strand from 5′-endof the antisense strand) were found to have a statistically significantimprovement in activity compared to the parent compound AD-57727.

In Vivo Evaluation

The target silencing of the siRNAs was assessed by qPCR. The performanceof the motifs was assessed to target mTTR. Animals (n=3/group) wereadministered a single siRNA dose of 3 mg/kg and liver levels weremeasured, first at pre-dose, and then at days 7 and 22, as shown in FIG.11.

FIG. 12 shows the enhanced activity with Stability Enhanced ConjugateChemistry (SEC-C), where the liver was assessed for activity (mRNA) onday 7 post-dosing. Animals received single dose of 3 mg/kg (s.c.). Thedata demonstrates the impact of the motifs on the in vivo activity.

FIG. 13 shows enhanced activity (approximately 4-fold improvement inactivity) with the new motifs (Motifs 1 and 2) compared to the parentcompound with data assessed on day 7 post-dosing. The data demonstratesthe impact of the motifs on the in vivo activity. The fold improvementis consistent across sequences.

FIG. 14 shows markedly improved duration across all three sequences,which demonstrate that the new motifs exhibit enhanced duration.

FIG. 15 shows the results of ApoC3-GalNAc3 SAR, at single 3 mg/kg SCdose hAAV 1×10¹¹ GC/mouse.

Example 5: VP and PS₂ Modifications at the 5′ End of the AntisenseStrand

Set forth below are exemplary protocols for the synthesis ofoligonucleotides containing 5′-vinyl phosphonate (VP) and the synthesisof oligonucleotides containing 2′-deoxythymidine linked via aphosphorodithioate (PS₂) linkage at the 5′-end of the oligonucleotide.One skilled in the art will appreciate that these same or similartechniques can be used to synthesize similar oligonucleotides. Othersynthesis techniques known to those of skill in the art may also be usedto synthesis and prepare these and similar oligonucleotides and themodifications, including, but not limited to, synthesis techniquesdisclosed in Whittaket et al., “Stereoselective synthesis of highlyfunctionalized P-stereogenic nucleosides via palladium-catalyzed P—Ccross-coupling reactions,” Tetrahedron Letters 49: 6984-87 (2008); Zhaoand Caruthers, “Synthesis and Preliminary Biochemical Studies with5′-Deoxy-5′-methylidyne Phosphonate Linked Thymidine Oligonucleotides,”Tetrahedron Letters 37(35): 6239-42 (1996); and U.S. Patent ApplicationPublication No. 2013/0084576, all of which are herein incorporated byreference in their entirety.

Protocols for Synthesis of Oligonucleotides Containing 5′-VinylPhosphonate

Introduction of Pivaloyloxymethyl-(POM)-Protected VP

Coupling and Oxidation:

Coupling of amidite was performed under standard synthesis conditionsusing 0.25M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation.Standard thiolation protocols with either 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide(PADS) were performed to convert the phosphite triester into aphosphorothioate linkage. Since the vinyl phosphonate building blockdoes not contain a DMT protecting group at the 5′-position, the finaldetritylation step was omitted.

Deprotection and Cleavage:

After synthesis the vinylphosphonate-containing oligonucleotides weredeprotected in a 3:1 mixture of aqueous NH₃ and EtOH with the additionof 1-2.5% by volume of 40% methylamine solution for 5 hours at 60° C. or16 hours at 35° C.

Introduction of Ethyl-Protected VP

Coupling and Oxidation:

Coupling of amidite was performed under standard synthesis conditionsusing 0.25M 5-(ethylthio)-1H-tetrazole in acetonitrile for activation.Standard thiolation protocols with either 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) or phenylacetyl disulfide(PADS) were performed to oxidize the phosphite triester and introducethe phosphorothioate linkage. Since the vinyl phosphonate building blockdoes not contain a DMT protecting group at the 5′-position, the finaldetritylation step was omitted.

Deprotection and Cleavage:

A solution of acetonitrile (ACN) and pyridine (Pyr) 50:1 (v/v) wasprepared and 3 Å molecular sieves were added to keep the mixture as dryas possible. To this mixture, 3.5 mL (5 g) of trimethylsilyl iodide(TMSI) was added for every 135 mL of the ACN/Pyr solution. This solutionmust be prepared fresh and has a maximum shelf life of one day. Next, a0.5 M solution of mercaptoethanol in 1:1 (v/v)acetonitrile-triethylamine was prepared and 3 Å molecular sieves wereadded. With the 5′-VP-containing oligonucleotide on resin and in thesynthesis column, the TMSI solution was slowly added at about 5-10 CVand allowed to react for 15 min. This step was repeated twice resultingin a total exposure time of approximately 45 minutes. Subsequently, theresin was washed extensively with ACN followed by a flow of themercaptoethanol solution of about 5-10 column volumes over the column,allowed to react for 10 min. This step was repeated once for a totalexposure of 20 minutes. After another extensive wash with ACN, thesupport-bound oligonucleotide was deprotected and cleaved from thesupport using standard conditions.

Protocol for Synthesis of Oligonucleotides Containing 2′-DexoythymidineLinked Via a Phosphorodithioate Linkage at the 5′-End of theOligonucleotide

Coupling and Oxidation:

the phosphoramidite solution was prepared from commercially availabledT-thiophosphoramidite (Glen Research) according to the manufacturer'sprotocol in dry acetonitrile at a concentration of 0.15 M. Coupling wasperformed under standard conditions using 0.25M5-(ethylthio)-1H-tetrazole in acetonitrile for a total coupling time of17 minutes. The capping step was omitted from the synthesis cycle.Oxidation (thiolation) was performed using 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) by extending reagent deliverand reaction times to 3×10 minutes. Final detritylation was performedusing standard synthesis conditions

Deprotection and Cleavage:

the solid support (on column) was washed with 0.5M piperidine in ACN(2×15 minutes exposure time) before the resin was transferred tosuitable container and treated under standard conditions (e.g. 3:1aqueous NH:EtOH solution for 5 hours at 60° C. or 16 hours at 35° C.) tocleave from solid support and deprotect the oligonucleotide.

The rest procedures for the oligonucleotide synthesis process aresimilar to the procedures described in Example 2.

FIG. 16 illustrates a schematic of Ago2 loaded siRNA. Generally,5′-phosphate-functionalized siRNAs (ESC chemistry) exhibit improved invitro activity. For instance, ˜80% of sequences tested have shownimproved inherent potency when transfected in vitro, and ˜30% show abouta 10-fold IC₅₀ benefit. In vivo, however, 5′-phosphate is rapidly lostin endo/lysome compartments. A modified phosphate, mimicking stablephosphate, the 5′-vinylphosphonate (5′-VP), is also shown in FIG. 16attached to the 5′ end of a modified oligonucleotide. This phosphonatewas originally designed by Merck.

An embodiment of this invention is directed towards 5′-end-modificationsfor potency improvements (RISC loading). The end modifications providestable phosphate mimics and promote endogenous phosphorylation.

FIG. 17 depicts a chart showing how the presence of 5′-VP generallyimproves in vivo activity, based on the evaluation of four differentApoB sequences. The LDL levels 7 days post single SC dose of 3 mg/kgwere analyzed for the four conjugates (with or without 5′-VPmodification). As seen from the chart, a 3-fold improvement in ED₅₀ isseen in certain ApoB sequences. The in vivo benefit has been confirmedwith additional compounds/targets including ApoC3, Tmpssr6, and TTR. TheApoB sequences are listed in Table 9.

TABLE 9 (Table 9 discloses SEQ ID NOS 191-206,respectively, in order of columns) Duplex Sense strand Antisense strandName (5′-3′) (5′3′) AD 63750 AfsasAfgAfgGfu usUfsuGfaAfgCfcGfUfAfuGfgCfuU AfuacAfcCfuCfuU fcAfaAfL96 fuscsa AD 64557 AfsasAfgAfgGfuVPusUfsuGfaAfgC GfUfAfuGfgCfuU fcAfuacAfcCfuCf fcAfaAfL96 uUfuscsaAD 63734 CfsusGfgAfcAfu usUfsuCfuUfgUfu UfCfAfgAfaCfaA CfugaAfuGfuCfcAfgAfaAfL96 fgsgsg AD 64560 CfsusGfgAfcAfu VPusUfsuCfuUfgU UfCfAfgAfaCfaAfuCfugaAfuGfuCf fgAfaAfL96 cAfgsgsg AD 63716 UfsgsUfgAfcAfausUfsgAfuGfcCfc AfUfAfuGfgGfcA AfuauUfuGfuCfaC fuCfaAfL96 fasasaAD 64559 UfsgsUfgAfcAfa VPusUfsgAfuGfcC AfUfAfuGfgGfcA fcAfuauUfuGfuCffuCfaAfL96 aCfasasa AD 63711 CfscsUfgGfaCfa usUfscUfuGfuUfcUfUfCfaGfaAfcA UfgaaUfgUfcCfaG faGfaAfL96 fgsgsu AD 64561 CfscsUfgGfaCfaVPusUfscUfuGfuU UfUfCfaGfaAfcA fcUfgaaUfgUfcCf faGfaAfL96 aGfgsgsu

FIG. 18 depicts different chemical modifications that can replace the PSlinkage, including phosphorodithioate (PS₂), and methylphosphonate(MePhos), which promotes endogenous phosphorylation. Modified siRNAs aregenerally not good substrates for Clp1 kinase, perhaps because of theinterference by 2′OMe modification at the first nucleotide of the ASstrand. However, the 2′-OMe modification along with phosphorothioatelinkage are desirable for exonuclease protection. Replacing the 2′-OMemodification with, for instance, 2′F, and modifying the PS linkage canpromote exonuclease protection while retaining metabolic stability.

FIG. 19 shows a chart of an in vitro evaluation of end modifications,including 2′-OMe-MePhos, 2′-OMe-PS, dN(PS₂), and 2′F-PS. As shown in thechart, the dn(PS₂) linkage and 2′F-PS showed improved in vitro activityrelative to the parent (2′OMe-PS). In particular, the dN(PS)₂ was stablein the in vitro tritosome assay, while the 2′F-PS showed metabolicliability. Transfections of primary mouse hepatocytes at 10 nM and 0.1nM (n=4) were carried out on two ApoB conjugates.

FIG. 20 shows two charts showing how a minor change at the antisense5′-end can significantly improve in vivo efficacy. FIG. 20A shows that2′F-PS at position 1 of the antisense strand can improve activity of5′P-dependent sequences, and FIG. 20B shows a ˜3-fold improved potencyby dN(PS)₂ over the parent, similar to 5′-VP.

Example 6: 5′-VP Modifications and Evaluation on siRNA ActivitySynthesis of 5′ Vinylphosphonate Phosphoramidite with PivaloxymethylProtecting Group

Reagents and Reaction Conditions for Scheme 1:

(a) Dess-Martin periodinane, DCM, 0° C.; (b) NaH,tetra(pivaloyloxymethyl) bisphosphonate, THF, −78° C., followed bystirring at 0° C., 70% (E and Z isomers); (c) formic acid: water, 1:1,24 hours, separated E and Z isomers by silica column chromatography orby RP-HPLC (reversed phase HPLC); (d) 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite, 5-(Ethylthio)-1H-tetrazole,ACN, 6 hours, room temperature, 65%.

Synthesis of tetra(pivaloyloxymethyl)-bis-phosphonate (X)

Tetramethyl methylenebisphosphonate (120 g, 0.51 mol), NaI (308 g, 2mol), chloromethyl pivalate (387 g, 2.5 mol) and acetonitrile (400 ml)were mixed and refluxed overnight. TLC (thin-layer chromatography) inEtOAc with 5% methanol confirmed the formation of product. The reactionmixture was diluted with ether (1000 ml) and washed with water (2×1000ml), dried with Na₂SO₃ and evaporated. The solid residue was washed withcold hexane and dried in vacuum to give 148 g (45%) of X as a paleyellow solid.

¹H NMR (500 MHz, CDCl₃): δ 5.73-5.63 (m, 8H), 2.65 (t, 2H), 1.22 (s,36H); ³¹P NMR (500 MHz, CDCl₃): δ 18.61.

Preparation of Compound 2

To an ice cold solution of compound 1 (3.0 g, 8 mmol) in 150 mL ofanhydrous dichloromethane was added Dess-Martin periodinane (DMP) (1.4equivalents; 4.7 g, 11.2 mmol). The reaction mixture was stirred at 0°C. for 1 hour and then at room temperature for 3 hours. TLC confirmedthe formation of product. The reaction mixture was then added to 200 mlsolution of 10% Na₂S₂O₃ and saturated NaHCO₃(1:1), followed by additionof 200 ml ethyl acetate. The crude aldehyde was extracted in ethylacetate dried and concentrated under reduced pressure. The crudealdehyde was used without any purification for next step.

Yield=2.87 gm (97%); purity by NMR approximately 70%; LC-MS: m/z 371.

Preparation of Compound 3

A solution of tetra polyoxometalate (POM)-bisphophonate sodium salt wasprepared by addition of bisphosphonate (X) in 14 ml THF (12.6 gm, 20mmol) to a suspension of NaH (0.58 g, 24 mmol) in 20 mL of THF at −78°C. and stirred for 15 minutes.

A solution of the aldehyde 2 (2.86 g) in 40 mL of anhydrous THF wasadded dropwise, to the above-prepared tetra (POM) bisphosphonate sodiumsalt solution at −78° C. The reaction mixture was stirred at −78° C. for1 hour, 0° C. for the next hour and then at room temperature for onehour. TLC confirmed the formation of product (EtOAc: hexane 7:3). Thecrude reaction mixture was added to 300 ml saturated ammonium chlorideand extracted with 300 ml ethyl acetate. The organic layer was washedwith brine, dried over sodium sulphate. The solution was thenconcentrated under reduced pressure and the residue was purified bysilica gel column chromatography (EtOAc in hexane=20-100%) to givecompound 3 (4.0 g) as a mixture of E/Z isomers (88/12) in 72% yield.

Preparation of Compound 4

A solution of 3 (4 g, 5.7 mmol) in 200 mL of HCOOH/H₂O (1:1, v: v) wasstirred at room temperature for 24 hours. TLC confirmed the formation ofproduct (MeOH:CH₂Cl₂=5:95).

The solution was concentrated under reduced pressure and the residue waspurified by silica gel column chromatography (MeOH:CH₂Cl₂=7:93 v/v).Fractions were tested on RP-HPLC (C18 column, buffer A=0.05% TFA inwater, buffer B=0.05% TFA in ACN; gradient 5-95% over 25 minutes) toconfirm the purity of two isomers (E and Z isomers): E isomer elutes at14.1 minutes and Z isomer elutes at 14.9 minutes. Initial fractions fromsilica gel chromatography contained mixture of E and Z isomers, and therest of the fractions were E isomer. The fractions containing mixture ofE and Z isomers were purified on RP-HPLC. Obtained 4-E isomer 2.3 g, 71%yield.

E Isomer:

¹H NMR (400 MHz, Acetonitrile-d₃): δ 8.98 (s, 1H), 7.30 (d, J=8.1 Hz,1H), 6.80 (ddd, J=23.7, 17.2, 5.0 Hz, 1H), 6.02 (ddd, J=21.6, 17.1, 1.7Hz, 1H), 5.77 (d, J=3.2 Hz, 1H), 5.57 (m, 5H), 4.32 (m, 1H), 4.01 (dd,J=7.0, 5.4 Hz, 1H), 3.82 (dd, J=5.5, 3.2 Hz, 1H), 3.41 (s, 3H), 1.14 (d,J=1.5 Hz, 18H); ³¹P NMR (162 MHz, Acetonitrile-d₃): δ 18.29.

Z Isomer:

¹H NMR (500 MHz, Acetonitrile-d₃): δ 9.50 (s, 1H), 7.44 (d, J=8.1 Hz,1H), 6.69 (ddd, J=54.4, 13.3, 8.7 Hz, 1H), 5.93 (ddd, J=17.8, 13.3, 1.3Hz, 1H), 5.80 (d, J=2.9 Hz, 1H), 5.69-5.58 (m, 5H), 5.22 (m, 1H), 4.01(dd, J=7.1, 5.3 Hz, 1H), 3.88 (dd, J=5.3, 2.9 Hz, 1H), 3.49 (s, 3H),1.19 (d, J=5.8 Hz, 18H); ³¹P NMR (202 MHz, Acetonitrile-d₃): δ 18.75.

Preparation of Compound 5

To a solution of compound 4-E isomer (2.1 g, 3.62 mmol) and ethylthiotetrazole (0.46 g, 3.62 mmol) in ACN (40 mL) was added 2-cyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite (1.311 g, 4.35 mmol). Themixture was stirred at room temperature for 2 hours. TLC in Hexane:EtOAc (2:8 in 0.15% TEA) confirmed the formation of product. Thereaction mixture was filtered, concentrated, and loaded onto a silicacolumn. The sample was eluted with 20% to 100% EtOAc in hexane with TEA(0.15%) to afford compound 5 as white foam (1.75 g, 62%).

E Isomer:

¹H NMR (400 MHz, Acetonitrile-d₃): δ 9.09 (s, 1H), 7.38 (d, J=8.1 Hz,1H), 6.89 (m, 1H), 6.10 (dddd, J=21.4, 17.1, 2.8, 1.7 Hz, 1H), 5.86 (t,J=3.8 Hz, 1H), 5.67-5.55 (m, 5H), 4.66-4.50 (m, 1H), 4.40-4.20 (m, 1H),3.99 (m, 1H), 3.92-3.57 (m, 4H), 3.44 (s, 3H), 2.73-2.64 (m, 2H), 2.14(s, 1H), 1.24-1.14 (m, 30H); ³¹P NMR (162 MHz, Acetonitrile-d₃): δ151.79 (d, J=71.3 Hz), 18.07 (d, J=54.0 Hz).

Z Isomer:

¹H NMR (400 MHz, Acetonitrile-d₃): δ 9.02 (s, 1H), 7.41 (dd, J=8.1, 1.6Hz, 1H), 6.62 (dddd, J=53.7, 13.1, 9.7, 7.0 Hz, 1H), 5.97 (dd, J=17.4,13.1 Hz, 1H), 5.80 (dd, J=7.0, 3.5 Hz, 1H), 5.70-5.52 (m, 5H), 5.41 (m,1H), 4.40-4.10 (m, 1H), 4.06-3.98 (m, 1H), 3.93-3.56 (m, 4H), 3.47 (s,3H), 2.68 (m, 2H), 2.14 (s, 1H), 1.33-1.11 (m, 30H); ³¹P NMR (202 MHz,Acetonitrile-d₃): δ 150.81 (d, J=141.4 Hz), 15.17.

Protocols for Synthesis of Oligonucleotides Containing 5′-VinylPhosphonate

The vinylphosphonate monomers and 5′-VP modified oligonucleotidesynthesis were done similar to the procedures in the literature (WO2008/100447 to Chen et al.; Lima et al. “Single-Stranded siRNAs ActivateRNAi in Animals,” Cell 150: 883-894 (2012); Prakash et al.,“Identification of metabolically stable 5-phosphate analogs that supportsingle-stranded siRNA activity,” Nucleic Acids Research 43: 2993-3011(2015), which are hereby incorporated by reference in their entirety).Briefly, the 5′-phosphate is protected by ethyl ether, and then, theethyl ether-protected phosphate goes through two-step deprotection: 1)TMS-I on solid support under anhydrous condition and 2) a standardoligonucleotide deprotection to obtain a 5′-VP modified oligonucleotide.This process is also discussed in Example 5.

Effect of Metabolically Stable (E) and (Z) 5′-Vinylphosphonate on siRNAActivity

Double-stranded small interfering RNA (siRNA) with 5′-phosphorylatedantisense strand facilitates efficient loading onto RNA-inducedsilencing complex (RISC) to elicit robust RNAi mediated gene silencing.Endogenous 5′-phosphorylation by Clp1 kinase of synthetic siRNAs is,therefore, critical for RISC loading and strand selection (Weitzer etal., “The human RNA kinase hClp1 is active on 3′ transfer RNA exons andshort interfering RNAs,” Nature 447: 222-226 (2007)). Phosphate mimicswith metabolically stable linkage have been used for nucleosidemodifications as antivirals (WO 2008/100447 to Chen et al.), for 5′-endmodification of siRNAs to improve gene silencing activity over thecorresponding non-phosphorylated siRNAs, in particular single strandedsiRNA (Lima et al. “Single-Stranded siRNAs Activate RNAi in Animals,”Cell 150: 883-894 (2012); Prakash et al., “Identification ofmetabolically stable 5-phosphate analogs that support single-strandedsiRNA activity,” Nucleic Acids Research 43: 2993-3011 (2015)).

In this example, the effect of phosphate mimics in double strandedsiRNAs were evaluated both in vitro and in vivo.

The siRNA sequences used in this example are shown in the tables below.The table discloses SEQ ID NOS 207-214, respectively, in order ofcolumns.

Duplex ID Sense Sequence Antisense Sequence AD-66572 usgsgaagCfaGfUfusCfscauCfaAfCfauac AfuguugauggaL96 UfgCfuuccasasa AD-68365.3usgsgaagCfaGfUf VPuCfcauCfaAfCfauac AfuguugauggaL96 UfgCfuuccasasaAD-68431.1 usgsgaagCfaGfUf VPUfCfcauCfaAfCfaua AfuguugauggaL96cUfgCfuuccasasa AD-68433.1 usgsgaagCfaGfUf VP(Tam)CfcauCfaAfCfAfuguugauggaL96 auacUfgCfuuccasasa u = 2′OMe, 5′ OH U Vpu = 2′OMe, 5′VPU VPUf = 2′F, 5′VP U VP(Tam) = 2′N-methylacetamide, 5′VP T

% knockdown of Modification at Factor IX @ 1 N1 of antisense mg/kg (day14) 2′OMe, 5′OH U 46 2′OMe, 5′VP U 80 2′F, 5′VP U 832′N-methylacetamide, 5′VP T 77

The impact of 5′-vinylphosphonate (VP) with E- and Z-geometry on doublestranded siRNA activity were compared. The results show that in vivoefficacy for chemically modified siRNAs can be improved with5′-trans-(E-)VP that mimics natural phosphate well, whereas,5′-cis-(Z-)VP did not show improvement in efficacy, suggesting that theZ-isomer does not mimic the natural phosphate well.

FIGS. 21A-B show the SAR analysis of in vitro and in vivo activity ofApoB siRNAs containing 5′-OH versus 5′-E-VP modification (at the 5′-endof the antisense strand). FIG. 21A shows the results with in vitrotransfection mouse 1° hepatocytes. FIG. 21B shows the LDL levels 3 dayspost a single dosing (SC dosing). The results of FIG. 21B demonstratethat ApoB siRNAs that were modified with 5′-E-VP showed improvedactivity.

FIG. 22 shows the results of in vitro potency of 5′-E-VP modificationversus 5′-Z-VP modification to mTTR and F9 siRNA-GalNAc conjugates. Theresults were from in vitro transfection mouse primary hepatocytes. Asshown in the figure, the siRNA conjugate that was modified with 5′-E-VPshowed retained or improved potency, whereas the siRNA conjugate thatwas modified with 5′-Z-VP showed decreased potency.

FIG. 23 shows the results of in vivo comparison of 5′-E-VP modificationversus 5′-Z-VP modification to F9 siRNA-GalNAc conjugate (single SCdosing). The results demonstrate that the siRNA conjugate that wasmodified with 5′-E-VP showed improved gene silencing activity over the5′-OH control, whereas the siRNA conjugate that was modified with5′-Z-VP showed a similar activity to that of the 5′-OH control.

The results of these figures show that 5′-phosphorylation of antisensestrand is desirable for efficient RNAi mediated gene silencing. Theefficacy of chemically modified siRNAs can be improved with5′-trans-vinylphosphonate (5′-E-VP) which mimics natural phosphate well.

Example 7: 5′-C-Malonyl Modifications and Evaluation on siRNA Activity

Synthesis and Incorporation of 5′-C-Malonyl Nucleotides to the 5′-End ofsiRNA:

General Experimental Conditions:

All moisture-sensitive reactions were carried under anhydrous conditionsunder argon atmosphere. Flash chromatography was performed on a TeledyneISCO (Lincoln, Nebr.) Combi Flash system using pre-packed ReadySepTeledyne ISCO silica gel columns. Electrospray ionization—highresolution mass spectrometry (ESI-HRMS) spectra were recorded on Waters(Milford, Mass.) Q-Tof API-US spectrometer using the direct flowinjection in the positive mode (capillary=3000 kV, cone=35, sourcetemperature=120° C., and desolvation temperature=350° C.). ¹H and ¹³CNMR spectra were recorded at room temperature on Varian spectrometers(Palo Alto, Calif.) at 400 MHz (¹H) and 126 MHz (¹³C), and chemicalshifts in ppm were referenced to the residual solvent peaks. Couplingconstants were given in Hertz. Signal splitting patterns were describedas singlet (s), doublet (d), triplet (t), quartet (q), broad signal(br), or multiplet (m). ³¹P NMR spectra were recorded at 162 MHz underproton-decoupled mode, and chemical shifts were referenced to externalH3PO4 (80%). LC/ESI-MS was performed on an Agilent (Santa Clara, Calif.)6130 single quadrupole LC/MS system using an XBridge C8 column (2.1×50mm, 2.5 μm) at 60° C. Buffer A consisted of 100 mM1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 16.3 mM triethylamine (TEA)in H₂O, and buffer B was 100% methanol.

Reagents and Conditions for Scheme 2:

(a) benzyloxymethyl acetal (BOM) chloride, DBU, DMF, 30 minutes, 0° C.,quantitative (Kurosu et al., “Synthetic studies towards theidentification of novel capuramycin analogs with mycobactericidalactivity,” Heterocycles 77: 217-225 (2009); Kurosu et al., “ConciseSynthesis of Capuramycin,” Org. Lett. 11:2393-2396 (2009), which areincorporated by reference in their entirety); (b)methyltriphenoxyphosphonium iodide, DMF, 15 minutes, room temperature,92%; (c) sodium methoxide, dimethyl malonate, 1,2-DME, 24 hours, reflux,92%; (d) 10% Pd/C, H₂ atm, i-PrOH/H₂O (10:1, v/v), 0.05 equivalentsformic acid, 12 hours, room temperature, 98% (Aleiwi et al., “A reliablePd-mediated hydrogenolytic deprotection of BOM group of uridine ureidonitrogen,” Tetrahedron Lett. 53: 3758-3762 (2012), which is incorporatedby reference in its entirety); (e) NEt₃-3HF, THF, 48 hours, roomtemperature, 88%; (d) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite,DIEA, DCM, 18 hours, room temperature, 56%; (g) 1 M aqueous piperidine,24 hours, room temperature; then 30% aqueous ammonia/ethanol (3:1, v/v),36 hours, room temperature, quantitative, Z⁺=piperidinium.

Synthesis ofN³-benzyloxymethyl-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine (2)

2′-O-Methyl-3′-O-tert-butyldimethylsilyl-uridine (1, 20 g, 53.7 mmol)was transformed in 2 (26.5 g, quantitative) following a variant of apreviously reported procedure.

Synthesis ofN³-benzyloxymethyl-5′-deoxy-5′-iodo-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine(3)

Compound 2 (10 g, 20.3 mmol) was dissolved in 100 mL of anhydrous DMF,and 20 g (40.6 mmol) of methyltriphenoxyphosphonium iodide were added.The mixture was stirred at room temperature for 15 minutes. Methanol(200 mL) was added to the reaction, and the mixture was stirred foradditional 15 minutes. The solvents were evaporated to dryness; theresidue was dissolved in dichloromethane (DCM) and washed with a 5%solution of Na₂S₂O₃ followed by water washing. The organic layers werecollected, dried over Na₂SO₄, filtered, and evaporated to dryness. Thecrude residue was purified by silica gel chromatography, using 0-50%ethyl acetate (EtOAc) in hexanes as eluent to obtain 3 as white foam(11.2 g, 92%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.77 (d, J=8.2 Hz, 1H), 7.30 (m, 5H), 5.90(d, J=5.2 Hz, 1H), 5.85 (d, J=8.2 Hz, 1H), 5.33 (d, J=13.0 Hz, 1H), 5.30(d, J=13.0 Hz, 1H), 4.58 (s, 2H), 4.23 (t, J=4.5 Hz, 1H), 4.07 (t, J=5.1Hz, 1H), 3.87 (q, J=6.1 Hz, 1H), 3.55 (dd, J=10.6, 6.3 Hz, 1H), 3.39(dd, J=10.6, 6.3 Hz, 1H), 3.32 (s, 3H), 0.89 (s, 9H), 0.14 (s, 3H), 0.12(s, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 161.7, 150.7, 140.2, 138.0,128.2, 127.4, 127.3, 101.6, 87.9, 83.3, 80.8, 72.7, 71.0, 70.1, 57.6,25.6, 17.7, 6.2, −4.7, −4.8.

HRMS-ESI: calculated for C₂₄H₃₅IN₂NaO₆Si (M+Na)⁺ is 625.1207; found:625.1205.

Synthesis ofN³-benzyloxymethyl-5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine(4)

Sodium methoxide (2 g, 33 mmol) was placed in a dry round-bottom flask,dimethyl malonate (12 mL, 100 mmol) and anhydrous 1,2-dimethoxyethane(DME, 100 mL) were added, and the mixture was brought to reflux.Compound 3 (10 g, 16.5 mmol), after being co-evaporated twice withanhydrous acetonitrile, was dissolved in 70 mL of anhydrous DME andadded to the refluxing solution of dimethyl malonate and sodiummethoxide. Reflux was continued for 24 hours. The reaction mixture wascooled to room temperature, and methanol (50 mL) was added to quench thereaction. Solvents and volatiles were evaporated in vacuo. The cruderesidue was purified by silica gel chromatography, using 0-100% EtOAc inhexanes as eluent to obtain compound 4 as colorless oil (9.2 g, 92%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.66 (d, J=8.2 Hz, 1H), 7.30 (m, 5H), 5.80(d, J=8.2 Hz, 1H), 5.76 (d, J=4.0 Hz, 1H), 5.33 (d, J=13.4 Hz, 1H), 5.30(d, J=13.4 Hz, 1H), 4.58 (s, 2H), 4.14 (t, J=5.4 Hz, 1H), 3.91 (m, 1H),3.76 (m, 1H), 3.64 (m, 4H), 3.60 (s, 3H), 3.33 (s, 3H), 2.37-2.09 (m,2H), 0.87 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H). ¹³C NMR (126 MHz,DMSO-d₆): δ 169.1, 168.8, 161.9, 150.6, 140.4, 138.0, 128.1, 127.4,127.3, 101.3, 88.6, 88.5, 81.3, 80.9, 73.1, 71.0, 70.0, 59.7, 57.5,52.5, 48.0, 31.5, 25.6, 17.7, −4.76, −5.06.

HRMS-ESI: calculated for C₂₉H₄₂N₂NaO₁₀Si (M+Na)⁺ is 629.2506; found:629.2508.

Synthesis of5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-methyl-3′-O-tert-butyldimethylsilyluridine(5)

Compound 4 (8.7 g, 14.3 mmol) was dissolved in 660 mL ofiso-propanol/water (10:1, v/v), and 0.9 g of 10% Pd/C was added,followed by 27 mL (0.7 mmol) of formic acid. The air from the flask wasremoved under vacuum; the reaction flask was flushed with hydrogen andwas stirred under hydrogen atmosphere at normal pressure at roomtemperature for 12 hours. The reaction mixture was filtered throughcelite and rinsed with ethanol. The filtrates were collected andevaporated to dryness. The crude residue was purified by silica gelchromatography, using 0-5% MeOH in DCM as eluent. The appropriatefractions were pooled and evaporated to dryness to obtain 5 as whitefoam (6.7 g, 98%).

¹H NMR (400 MHz, DMSO-d₆): δ 11.38 (d, J=1.8 Hz, 1H), 7.61 (d, J=8.1 Hz,1H), 5.71 (d, J=4.3 Hz, 1H), 5.65 (dd, J=8.0 Hz, J=2.1 Hz, 1H), 4.16 (t,J=5.3 Hz, 1H), 3.91 (t, J=4.8 Hz, 1H), 3.73 (m, 1H), 3.63 (m, 4H), 3.61(s, 3H), 3.31 (s, 3H), 2.24-2.07 (m, 2H), 0.87 (s, 9H), 0.08 (s, 3H),0.08 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆): δ 169.2, 168.9, 163.0, 150.4,141.2, 141.2, 102.1, 87.7, 81.2, 80.9, 73.1, 57.5, 52.5, 52.4, 52.3,48.0, 31.6, 25.6, 17.7, −4.8, −5.1.

HRMS-ESI: calculated for C₂₁H₃₄N₂NaO₉Si (M+Na)⁺ is 509.1931; found:509.1929.

Synthesis of 5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-methyluridine (6)

Compound 5 (6.7 g, 13.8 mmol) was stirred withtriethylamine-trihydrofluoride (11 mL, 202.5 mmol) in 150 mL ofanhydrous THF in a round-bottom flask at room temperature for 48 hours.The solvents were evaporated in vacuo to two-thirds the original volume.The residue was purified by silica gel chromatography, using 0-10% MeOHin DCM as eluent. The appropriate fractions were pooled and evaporatedto dryness to obtain 6 as white foam (4.5 g, 88%).

¹H NMR (400 MHz, DMSO-d₆): δ 11.37 (s, 1H), 7.56 (d, J=8.1 Hz, 1H), 5.72(d, J=4.3 Hz, 1H), 5.64 (d, J=8.1 Hz, 1H), 5.24 (d, J=6.3 Hz, 1H), 3.94(q, J=5.7 Hz, 1H), 3.86 (t, J=4.8 Hz, 1H), 3.72 (m, 1H), 3.64 (m, 4H),3.61 (m, 3H), 3.34 (s, 3H), 2.25-2.07 (m, 2H). ¹³C NMR (126 MHz,DMSO-d₆): δ 169.2, 169.0, 163.0, 150.3, 141.0, 102.0, 87.4, 81.6, 80.8,71.9, 57.6, 52.5, 52.4, 48.0, 31.9.

HRMS-ESI: calculated for C₁₅H₂₀N₂NaO₉ (M+Na)⁺ is 395.1067; found:395.1070.

Synthesis of5′-deoxy-5′-C-(dimethylmalonyl)-2′-O-methyluridine-3′-O—(O-(2-cyanoethyl)-N,N-di-isopropyl)phosphoramidite(7)

Compound 6 (3.0 g, 8 mmol) was co-evaporated three times with anhydrousacetonitrile and then dried overnight under vacuum over P205. The dryresidue was dissolved in 60 mL of anhydrous DCM; diisopropylethylamine(4.5 mL, 24 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite(2.2 mL, 10.0 mmol) were added successively. After 1 hour of stirringunder argon atmosphere, another 1.0 mL (4.0 mmol) of 2-cyanoethylN,N-diisopropylchlorophosphoramidite was added, and the stirringcontinued for additional 18 hours. The reaction mixture was diluted with150 mL of DCM and washed with 200 mL of saturated sodium bicarbonatesolution. The organic layer was dried with sodium sulfate and removed byfiltration. The solvents were evaporated in vacuo, and the crude residuewas purified by silica gel chromatography. The eluent washexanes/EtOAc/NEt₃, (66:33:1, v/v/v in a step gradient tohexanes/EtOAc/NEt₃ 33:66:1, v/v/v). The appropriate fractions werepooled, evaporated to dryness, co-evaporated with anhydrousacetonitrile, and dried under high vacuum to obtain 7 as white foam (3.2g, 56%).

¹H NMR (400 MHz, CD₃CN, mixture of diastereoisomers): δ 8.97 (s, 1H),7.36 (m, 1H), 5.78 (d, J=4.2 Hz, 1H), 5.64 (d, J=8.1 Hz, 1H), 4.23-3.80(m, 4H), 3.77-3.59 (m, 8H), 3.45-3.41 (m, 3H), 2.68 (t, J=5.9 Hz, 2H),2.44-2.31 (m, 2H), 1.42-1.00 (m, 12H). ³¹P NMR (162 MHz, CD₃CN, mixtureof diastereoisomers): δ 151.8, 151.6. ¹³C NMR (126 MHz, CD₃CN, mixtureof diastereoisomers): δ 170.6, 170.2, 163.8, 151.3, 141.3, 119.6, 103.0,102.9, 89.6, 89.2, 82.9, 82.5, 82.4, 81.8, 81.3, 81.2, 75.3, 75.2, 75.1,75.0, 59.8, 59.7, 59.3, 59.1, 58.9, 58.8, 53.3, 53.2, 49.5, 49.4, 44.2,44.15, 44.1, 44.0, 33.0, 25.0, 24.9, 24.8, 21.0, 20.9.

HRMS-ESI: calculated for C₂₄H₃₈N₄O₁₀P (M+H)⁺ is 573.2326; found:573.2321.

Synthesis of 5′-deoxy-5′-C-malonyl-2′-O-methyluridine, Piperidinium Salt(8)

Compound 6 (0.1 g, 0.3 mmol) was stirred with 1 M aqueous piperidine (10mL, 10 mmol) at room temperature for 24 hours. The solvents wereevaporated in vacuo, and the residue was dissolved in a mixture of 30%ammonia/ethanol (3:1, v/v) and stirred at room temperature for 36 hours.The solvents were evaporated in vacuo and 8 was obtained as colorlessoil (quantitative).

¹H NMR (400 MHz, D₂O): δ 7.75 (d, J=8.1 Hz, 1H), 5.92 (m, 2H), 4.16 (t,J=5.5 Hz, 1H), 4.06 (t, J=4.7 Hz, 1H), 3.99 (m, 1H), 3.50 (s, 3H), 3.27(t, J=7.0 Hz, 1H), 3.17 (t, J=5.7 Hz, 6H), 2.27-2.06 (m, 2H), 1.87-1.54(m, 8H). ¹³C NMR (126 MHz, D₂O): δ 179.5, 179.2, 168.0, 153.0, 142.5,103.1, 88.5, 83.7, 83.3, 72.7, 58.9, 55.9, 45.3, 34.7, 23.0, 22.2.

HRMS-ESI (M+H)⁺: calculated for C₁₃H₁₇N₂O₉ is 345.0929; found: 345.0919.

Oligonucleotide Synthesis

Oligonucleotides were synthesized on an ABI-394 DNA/RNA synthesizerusing modified synthesis cycles based on those provided with theinstrument. A solution of 0.25 M 5-(S-ethylthio)-1H-tetrazole inacetonitrile was used as the activator. The phosphoramidite solutionswere 0.15 M in anhydrous acetonitrile. The oxidizing reagent was 0.02 MI₂ in THF/pyridine/H₂O.N,N-Dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methanimidamide(DDTT), 0.1 M in pyridine, was used as the sulfurizing reagent. Thedetritylation reagent was 3% dichloroacetic acid (DCA) in DCM. In thecase of 5′-phosphate compounds, the Glen Research chemicalphosphorylation reagent (Cat. #10-1902-02) was used for the introductionof the 5′-monophosphate. After completion of the automated synthesis,the solid support was washed with 0.1 M piperidine in acetonitrile for10 minutes, then washed with anhydrous acetonitrile and dried withargon. The oligonucleotide was then manually released from support anddeprotected using a mixture of 30% NH₄OH/ethanol (3:1, v/v) or 40%methylamine (0.5 mL/μmol of solid support) for 6 hours at 55° C. or 15minutes at 60° C., respectively. Solvent was collected by filtration andthe support was rinsed with DMSO (1.5 mL/μmol of solid support).

The 5′-C-malonyl solid-supported oligonucleotides were first treatedwith 1 M aqueous piperidine (1.5 mL/μmol of solid support) for 24 hoursat room temperature, and the solution was filtered off and evaporated todryness. The residue was dissolved in a mixture of 30% NH₄OH/ethanol(3:1, v/v, 2 mL/μmol of solid support) and shaken at room temperaturefor 36 hours, then evaporated to dryness. Crude oligonucleotides werepurified by anion-exchange HPLC using a linear gradient of 0.22 M to0.42 M NaClO₄ in 0.02 M Tris-HCl, pH 8.5/50% (v) acetonitrile in 120-150minutes at room temperature. All single strands were purified to >85%HPLC (260 nm) purity and then desalted by size exclusion chromatographyusing an AP-2 glass column (20×300 mm, Waters) custom-packed withSephadex G25 (GE Healthcare), eluted with sterile nuclease-free water.Hybridization to generate siRNA duplexes was performed by mixingequimolar amounts of complementary strands to a final concentration of20 μM in 1×PBS buffer, pH 7.4, and by heating in a water bath at 95° C.for 5 minutes followed by slow cooling to room temperature.

Evaluation of 5′-C-Malonyl Modifications on Gene Silencing Activity andStability

5′-phosphorylation of double-stranded RNA is desirable for efficientloading of small interfering RNAs (siRNAs) into the RNA-inducedsilencing complex (RISC) resulting in RNAi-mediated gene silencing.Endogenous or exogenous siRNAs are generally readily phosphorylated by acytosolic kinase, and, in most cases, the presence of a synthetic5′-monophosphate is not required. However, in certain cases ofchemically modified siRNAs, metabolically stable 5′-phosphate mimics canlead to higher stability, increased RISC loading and more potent genesilencing.

In this example, the effect of a 5′-C-malonyl moiety, which wasincorporated as a modified nucleotide at the 5′-terminus of theantisense strand of chemically modified siRNAs using solid-phasesynthesis, was evaluated. The 5′-C-malonyl can exist as a di-anion atphysiological pH similar to the 5′-monophosphate di-anion. The in vitrogene silencing activity, metabolic stability and RISC loading of siRNAscontaining the 5′-C-malonyl group on the antisense strands wereevaluated and compared to the corresponding 5′-phosphorylated andnon-phosphorylated counterparts.

Cell Culture and Transfection

Primary mouse hepatocytes were obtained from Life Technologies andcultured in

Williams E Medium with 10% fetal bovine serum (FBS). Transfection wascarried out by adding 4.9 μL of Opti-MEM plus 0.1 μL of LipofectamineRNAiMax (Invitrogen) per well to 5 μL of each siRNA duplex at thedesired concentration to an individual well in a 384-well plate. Themixture was incubated at room temperature for 20 minutes and 40 μL ofcomplete growth media containing 5,000 cells was added to the siRNAmixture. Cells were incubated for 24 hours prior to RNA isolation. Asimilar procedure was followed for the transfection of 10,000,000 cells.Dose response experiments were done using eight 6-fold serial dilutionsover the range of 20 nM to 75 pM or 50 nM to 187.5 pM.

RNA Isolation

RNA was isolated using Dynabeads mRNA Isolation Kit (Invitrogen). Cellswere lysed in 75 μL of Lysis/Binding Buffer containing 3 μL of beads perwell and mixed for 10 minutes on an electrostatic shaker. Buffers wereprepared according to the manufacturer's protocol. The washing stepswere automated on a Biotek EL406 using a magnetic plate support. Beadswere washed (90 μL) once in Buffer A, once in Buffer B, and twice inBuffer E, with aspiration steps between washes.

cDNA Synthesis

cDNA synthesis was accomplished with the ABI High capacity cDNA reversetranscription kit (Applied Biosystems). A mixture of 1 μL 10× Buffer,0.4 μL 25×dNTPs, 1 μL random primers, 0.5 μL reverse transcriptase, 0.5μL RNase inhibitor, and 6.6 μL of water per reaction were added perwell. Plates were sealed, agitated for 10 minutes on an electrostaticshaker, and then incubated at 37° C. for 2 hours. Following this, theplates were agitated at 80° C. for 8 minutes.

Real-Time PCR

cDNA (2 μL) was added to a master mix containing 0.5 μL mouse GAPDHTaqMan Probe (Applied Biosystems, Cat. #4308313), 0.5 μL mouse ApoB orPTEN TaqMan probes (Applied Biosystems, Cat. #Mm01545156 ml andMm01212532 ml, respectively), and 5 Lightcycler 480 probe master mix(Roche) per well in a 384-well 50 plates (Roche). Real-time PCR was donein an ABI 7900HT RT-PCR system (Applied Biosystems) using the ΔΔCt (RQ)assay. Each duplex and concentration was tested in four biologicalreplicates. To calculate relative fold change, real time data wereanalyzed using the ΔΔCt method and normalized to assays performed withcells transfected with 10 nM non-specific siRNA. IC₅₀ values werecalculated using a 4-parameter fit model using XLFit.

TABLE 10 IC₅₀ values for 5′-C-malonyl, 5′-phosphate and5′-OH siRNAs in PTEN and ApoB silencing in cell-basedassays. Table 10 discloses SEQ ID NOS 215-232,respectively, in order of columns. 5′- siRNA modifi- sense antisenseIC₅₀ target cation strand (5′-3′)^(a) strand (5′-3′)^(a) (nM)^(b) 1 OHC•c•UgGaCaUUCaG u•U•cUuGuUcUga 1.0 ApoB aAcAaGaAGalNAc aUgUcCaGg•g•u 2phosphate C•c•UgGaCaUUCaG Pu•U•cUuGuUcUg 0.1 ApoB aAcAaGaAGalNAcaaUgUcCaGg•g•u 3 OH U•g•UgAcAaAUAuG u•U•gAuGcCcAua 0.5 ApoBgGcAuCaAGalNAc uUuGuCaCa•a•a 4 phosphate U•g•UgAcAaAUAuG Pu•U•gAuGcCcAu0.1 ApoB gGcAuCaAGalNAc auUuGuCaCa•a•a 5 malonate C•c•UgGaCaUUCaGMu•U•cUuGuUcUg 0.7 ApoB aAcAaGaAGalNAc aaUgUcCaGg•g•u 6 malonateU•g•UgAcAaAUAuG Mu•U•gAuGcCcAu 0.4 ApoB gGcAuCaAGalNAc auUuGuCaCa•a•a 7OH AaGuAaGgAcCaGaG uUgUcUcUgGuCcU 0.7 PTEN aCaAdT•dT uAcUudT•dT 8phosphate AaGuAaGgAcCaGaG PuUgUcUcUgGuCc 0.2 PTEN aCaAdT•dT UuAcUudT•dT9 malonate AaGuAaGgAcCaGaG MuUgUcUcUgGuCc 0.2 PTEN aCaAdT•dT UuAcUudT•dTNote: ^(a) P indicates 5′-monophosphate; M indicates 5′-malonate (i.e.,5′-C-malonyl); italicized upper case and normal lower case lettersindicate 2′-deoxy-2′-fluoro (2′-F), and 2′-O-methyl (2′-OMe) sugarmodifications, respectively; • indicates phosphorothioate (PS) linkage;dT indicates 2′-deoxythymidine nucleotide; GalNAc indicateshydroxyprolynyl tri-valent N-acetyl-galactosamine ligand (Nair et al.,“Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes inHepatocytes and Elicits Robust RNAi-Mediated Gene Silencing,” J. Am.Chem. Soc. 136, 16958-16961 (2014), which is incorporated by referencein its entirety). ^(b)half maximal inhibitory concentration (IC₅₀) forgene silencing in primary mouse hepatocytes. All values are fromtriplicate experiments.

Tritosome Stability Assay

Rat liver tritosomes (Xenotech, custom product PR14044) were thawed toroom temperature and diluted to 0.5 units/mL in 20 mM sodium citratebuffer, pH 5.0. Samples were prepared by mixing 100 μL of 0.5 units/mLacid phosphatase tritosomes with 25 μL of 0.4 mg/mL siRNA in amicrocentrifuge tube. After incubation for 4 hours or 24 hours in anEppendorf Thermomixer set to 37° C. and 300 rpm, 300 μL of PhenomenexLysis Loading Buffer and 12.5 μL of a 0.4 mg/mL internal standard siRNAwere added to each sample. Samples for time 0 were prepared by mixing100 μL of 0.5 units/mL acid phosphatase Tritosomes with 25 μL of 0.4mg/mL siRNA sample, 300 μL of Phenomenex Lysis Loading Buffer, and 12.5μL of a 0.4 mg/mL internal standard siRNA. siRNA was extracted from eachtime point sample (0 hour, 4 hours, 24 hours) using a Phenomenex ClarityOTX Starter Kit. The samples were then re-suspended with 500 μL ofnuclease free water, and 50 μL of sample was analyzed by LC/MS.

RISC Immunoprecipitation and RT-PCR Assay

siRNA-transfected primary mouse hepatocytes (10,000,000 cells) werelysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40,0.1% SDS) with protease inhibitor (Sigma-Aldrich). Lysate concentrationwas measured with a protein BCA kit (Thermo Scientific). For eachreaction, 2 mg of total lysate was used. Anti-Ago2 antibody waspurchased from Wako Chemicals (Clone No.: 2D4). Control mouse IgG wasfrom Santa Cruz Biotechnology (sc-2025). Dynabeads (Life Technologies)were used to precipitate antibodies. Ago2-associated siRNA andendogenous miR122 were measured by Stem-Loop RT followed by TaqMan PCRanalysis based on previously published methods.

Computational Simulation of the Interaction Between5′-deoxy-5′-C-malonyluridine and the Human Ago2 MID Domain

The recognition modes of available crystal structures of complexesbetween hAgo2 MID (amino acids 432-578; residues 440-572 are resolved inthe electron density) and UMP (PDB ID code 3LUJ) and full-length hAgo2and miR-20a (PDB ID code 4F3T) revealed that the recognition of5′-terminal phosphates are very similar. The only difference between thetwo structures is that in the complex with full-length Ago2, a residuefrom the PIWI domain (Arg-812) makes a contribution to the recognitionof the 5′-phosphate. The UMP:MID complex was therefore used as the basisfor modeling the interaction between 5′-malonyluridine and the hAgo2 MIDdomain. Three-dimensional coordinates of the UMP:MID complex wereretrieved from the Protein Data Bank (http://www.rcsb.org). Using theprogram UCSF Chimera (version 1.5.3), all water molecules from thecrystal structure were deleted and the 5′-phosphate group was convertedto the 5′-C-malonyl moiety. Hydrogen atoms were then added and thegeometry of 5′-deoxy-5′-C-malonyluridine and its orientation andH-bonding/non-bonded interactions at the 5′-phosphate pocket of thehAgo2 MID domain optimized with the Amber force field (ff12SB andGasteiger charges for standard amino acids and non-standard residues,respectively), as implemented in UCSF Chimera.

FIGS. 24A-C are graphs showing the dose-response curves for (A) 5′-OH,(B) 5′-C-malonyl, and (C) 5′-phosphate PTEN siRNAs in primary mousehepatocytes in an in vitro PTEN silencing assay. All values are fromtriplicate experiments.

FIG. 25 shows the results of enzymatic stabilities of 5′-OH,5′-C-malonyl, and 5′-phosphate siRNAs incubated in in rat livertritosomes. The siRNA target sequences are shown in Table 10. The siRNAswere incubated at 0.4 mg/mL (approximately 5 mM) concentration for 4hours and 24 hours, respectively, in the presence of tritosomes. Percentfull-length strand was determined by HPLC. The data were normalized tountreated controls.

FIG. 26 shows the results of RISC loading of 5′-OH, 5′-C-malonyl, and5′-phosphate siRNAs (5′-modification on the antisense strands)determined by immunoprecipitation of Ago2 from primary mouse hepatocytesand by RT-PCR amplification of the Ago2-loaded single strands. Levels ofendogenous miR122 were determined as a control. The siRNA targetsequences are shown in Table 10. siRNAs 7, 8, and 9 were transfectedinto cells at 10 nM. Levels of antisense strands of are given in nMsiRNA strand per mg of cell lysate.

The results of these figures show that the 5′-C-malonyl siRNAs sustainedor improved in vitro gene silencing, high levels of Ago2 loading, andconferred dramatically improved metabolic stability to the antisensestrand of siRNA duplexes, as compared to the corresponding5′-phosphorylated and non-phosphorylated counterparts. In silicomodeling studies showed favorable fit of the 5′-C-malonyl group withinthe 5′-phosphate binding pocket of hAgo2 MID. Therefore, the5′-C-malonyl, a metabolically stable 5′-phosphate bioisostere, hasexcellent biomimetic properties for use in therapeutic siRNAs.

Example 8: Process for Stereoselective Synthesis of 5′-C-AlkylNucleosides Using Trialkylaluminum or Dialkylzinc General Scheme forSynthesis of 5′-C-Alkyl Nucleosides by Addition of Mild AlkylNucleophiles to 5′-Nucleoside Aldehydes

Alternatively, instead of AlR′₃ or ZnR′₂ (listed above in Scheme 2),SnR′₄, TiR′₄, and various other metals, with the exception of Li and Mg,can be used with the R′ group in this reaction scheme.

General Scheme for Stereospecific Interconversion of Epimers of 5′-AlkylNucleosides Through Anhydro-Ring Closure

Synthesis of Pyrimidine 5′-Aldehydes 2a-d Using Dess-Martin Periodinane

5′-Deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-oxo-uridine2a. Dess-Martin periodinane (40.7 g, 96 mmol) was added to a stirred andcooled (0° C.) solution of 3′-OTBS protected uridine 1a (29.8 g, 80mmol) in anhydrous DCM (600 mL) under argon atmosphere. The cooling bathwas removed and the mixture was stirred at room temperature for 4 hours,after which time no starting alcohol 1a could be observed by TLC. Themixture was cooled to 0° C. and poured to a vigorously stirred mixtureof 10% solution of sodium thiosulfate (250 mL) and saturated solution ofsodium bicarbonate (350 mL). After stirring at room temperature for 45minutes significant precipitation occurred. The precipitate was filteredoff and the solids where washed with DCM (200 mL×2). The filtrate wasplaced in a separatory funnel, the organic phase was separated and driedover anhydrous sodium sulfate. The solids from the filter funnel weretransferred to an Erlenmeyer flask, acetone (450 mL) was added, thesuspension was stirred for 15 minutes, filtered, and the solids werewashed with acetone (200 mL×2). The acetone extract was evaporated, theresidue was combined with DCM extract, the solvent was evaporated, theresidue was dissolved in ACN-acetone 1:1 mixture (200 mL), the solventwas evaporated again, and the solid residue was dried in vacuum toafford crude aldehyde 2a 27.6 g (93%). Aldehyde content approximately71% (determined by CHO/NH ratio of H¹ NMR in ACN-d₃) that was used inthe next step without of further purification. The product could bestored without of notable decomposition at −20° C. under argonatmosphere. ¹H NMR of major component (400 MHz, ACN-d₃): δ 0.15 (s, 6H);0.93 (s, 9H); 3.37 (s, 3H); 3.62-3.68 (m, 2H); 3.81 (dd, J=4.6, 5.9 Hz,1H); 4.48 (d, J=3.4 Hz, 1H); 4.59 (ddd, J=0.4, 3.4, 4.5 Hz, 1H); 5.70(d, J=8.2 Hz, 1H); 5.94 (d, J=6.0 Hz, 1H); 7.71 (d, J=8.2 Hz, 1H); 9.17(bs, 1H); 9.68 (d, J=0.5 Hz, 1H).

2′,5′-Dideoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-fluoro-5′-oxo-uridine2b was prepared analogously from 1b (13.8 g, 38 mmol) and DMP (19.5 g,46 mmol) in anhydrous DCM (550 mL). After stirring overnight at roomtemperature, the mixture was cooled, quenched, and extracted with DCM toafford 12.7 g (93%) of crude aldehyde containing approximately 60% ofthe title product 2b. ¹H NMR of major component (400 MHz, ACN-d₃): δ0.13 (s, 3H); 0.14 (s, 3H); 0.92 (s, 9H); 4.41 (d, J=6.0 Hz, 1H); 4.67(ddd, J=4.9, 6.0, 13.6 Hz, 1H); 5.15 (ddd, J=2.8, 4.9, 52.5 Hz, 1H);5.68 (d, J=8.1 Hz, 1H); 5.89 (dd, J=2.8, 18.3 Hz, 1H); 7.55 (d, J=8.1Hz, 1H); 9.26 (bs, 1H); 9.64 (d, J=1.0 Hz, 1H).

5′-Deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-5′-oxo-thymidine 2c wasprepared analogously from 1c (17.9 g, 50 mmol) and DMP (25.4 g, 60 mmol)in anhydrous DCM (500 mL). After stirring for 3 hours at 0° C., themixture was quenched and extracted with DCM to afford 20.0 g (quant.) ofcrude aldehyde containing approximately 63% of the title product 2c. ¹HNMR of major component (400 MHz, ACN-d₃): δ 0.135 (s, 3H); 0.140 (s,3H); 0.92 (s, 9H); 1.85 (d, J=1.2 Hz, 3H); 2.08-2.24 (m, 2H); 4.38 (d,J=2.2 Hz, 1H); 4.75 (dt, J=2.2, 5.7 Hz, 1H); 6.24 (dd, J=6.2, 7.9 Hz,1H); 7.55 (d, J=1.3 Hz, 1H); 9.18 (bs, 1H); 9.65 (d, J=0.5 Hz, 1H).

N-Acetyl-5′-deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-oxo-cytidine2d was prepared analogously from 1d (33.1 g, 80 mmol) and DMP (40.7 g,96 mmol) in anhydrous DCM (600 mL). After stirring at room temperaturefor 4 hours, the mixture was quenched and treated analogously to thecase of 2a to afford crude aldehyde 33.2 g (100%) containingapproximately 56% of the title product 2d. ¹H NMR of major component(400 MHz, ACN-d₃): δ 0.108 (s, 3H); 0.117 (s, 3H); 0.92 (s, 9H); 2.14(s, 3H); 3.44 (s, 3H); 3.90-3.94 (m, 1H); 4.49-4.52 (m, 2H); 5.92 (d,J=3.8 Hz, 1H); 7.33 (d, J=7.5 Hz, 1H); 8.10 (d, J=7.5 Hz, 1H); 9.13 (bs,1H); 9.72 (s, 1H).

Synthesis of Purine 5′-Aldehydes 2e-f Using Dess-Martin Periodinane,“Water-Free Quench”

N-Benzoyl-5′-deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-oxo-adenosine2e, Dess-Martin periodinane (20.4 g, 48 mmol) was added to a stirred andcooled (0° C.) solution of 3′-OTBS protected adenosine 1e (20.0 g, 40mmol) in anhydrous DCM (300 mL) under argon atmosphere. The cooling bathwas removed and the mixture was stirred at room temperature for 3 hours,after which time no starting alcohol 1e could be observed by TLC.Isopropyl alcohol (0.61 mL, 8 mmol) was added and the stirring wascontinued for an additional 1 hour. The solvent was removed in vacuum,and ethyl acetate (220 mL) was added followed by slow addition ofhexanes (150 mL) over 4 hours with stirring. The mixture was stirred atroom temperature overnight, filtered, and the solids were washed twicewith ethyl acetate-hexanes (1:1) mixture. The filtrate was evaporated invacuum and the residue was co-evaporated with dry acetonitrile (300 mL).Acetonitrile (100 mL) was added to form a suspension that was stirredovernight, filtered, and the solids were washed twice with acetonitrile.The filtrate was evaporated in vacuum to afford white foamy residue thatwas dried under high vacuum to afford 20.2 g (100%) of crude aldehydecontaining approximately 54% of the title product 2e, that was used inthe next step without of further purification. ¹H NMR of major component(400 MHz, ACN-d₃): δ 0.188 (s, 3H); 0.190 (s, 3H); 0.97 (s, 9H); 3.34(s, 3H); 4.49 (dd, J=4.3, 6.3 Hz, 1H); 4.51 (dd, J=1.0, 2.9 Hz, 1H);4.90 (dd, J=3.0, 4.2 Hz, 1H); 6.24 (d, J=6.3 Hz, 1H); 7.54 (t, J=7.3 Hz,2H); 7.61-7.67 (m, 1H); 7.97-8.03 (m, 2H); 8.44 (s, 1H); 8.66 (s, 1H);9.50 (bs, 1H); 9.82 (d, J=1.0 Hz, 1H).

N-Isobuturyl-5′-deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-5′-oxo-guanosine2f was prepared analogously from if (19.3 g, 40 mmol) and DMP (20.4 g,48 mmol) in anhydrous DCM (300 mL). After stirring for 3 hours at roomtemperature, isopropyl alcohol (0.61 mL, 8 mmol) was added, and thestirring was continued for an additional 1 hour. The solvent was removedin vacuum, and ethyl acetate (225 mL) was added followed by slowaddition of hexanes (150 mL) over 30 minutes with stirring. The mixturewas stirred at room temperature for 5 hours, filtered, and the solidswere washed twice with ethyl acetate-hexanes (1:1.5) mixture. Thefiltrate was evaporated in vacuum and the residue was co-evaporated witha mixture of toluene (250 mL) and dry acetonitrile (250 mL) followed byacetonitrile (250 mL). The white foamy residue was dried in high vacuumto afford 21.5 g of crude aldehyde containing approximately 53% of thetitle product 2f, that was used in the next step without of furtherpurification. ¹H NMR of major component (400 MHz, ACN-d₃): δ 0.167 (s,3H); 0.175 (s, 3H); 0.95 (s, 9H); 1.18 (d, J=6.8 Hz, 3H); 1.19 (d, J=6.8Hz, 3H); 2.66-2.77 (m, 1H); 3.31 (s, 3H); 4.31 (dd, J=4.3, 7.0 Hz, 1H);4.48 (dd, J=1.0, 2.3 Hz, 1H); 4.69 (ddd, J=0.4, 2.4, 4.3 Hz, 1H); 6.01(d, J=7.0 Hz, 1H); 8.03 (s, 1H); 9.45 (s, 1H); 9.79 (d, J=1.1 Hz, 1H);12.05 (bs, 1H).

Stereoselective Addition of Mild Me-Nucleophiles to Nucleoside5′-Aldehydes.

a. General Observations.

Nucleobase-Dependence of Stereoselectivity:

As shown in the tables below, (S)-epimers of 5′-Me pyrimidinenucleosides can be synthesized with high level of stereoselectivityusing trimethylaluminum (as shown in the table for AlMe₃), whereas(S)-epimers of 5′-Me purines can be synthesized stereoselectively usingdimethylzinc (as shown in the table for ZnMe₂).

AIMe₃ (S:R) Solvent/T (° C.) U C^(Ac) A^(Bz) G^(i-Bu) THF (0-rt) 12:1  9:1 1:1 Slow 1:2 DCM (−78-rt) 3.9:1  2:1 1:1 Toluene (−78-rt) 3.4:1  3:1

ZnMe₂ (S:R) Solvent/T (° C) U C^(Ac) A^(Bz) G^(i-Bu) THF (0-rt) NR Slow2:1 DCM/Toluene Very 9:1 3:1 (−78-rt) slow

Solvent-Dependence of Stereoselectivity:

As shown in the tables, (S)-epimers of 5′-Me pyrimidine nucleosides canbe synthesized with high level of stereoselectivity usingtrimethylaluminum in THF (as shown in the tables for AlMe₃), whereas(S)-epimers of 5′-Me purines can be synthesized stereoselectively usingdimethylzinc in a non-coordinating solvent (as shown in the tables forZnMe₂). The equimolar mixture of purine stereoisomers can be obtainedwith trimethylaluminum either in THF (for A derivatives), or in anon-coordinating solvent (DCM) (for G-derivatives).

AIMe₃ (S:R) Dielectric Solvent/T (° C.) A^(Bz) U Constant (ε) Toluene(−78-rt) 3:1 3.4:1   2.38 DCM (−78-rt) 2:1 3.9:1   8.93 Dioxane 1.9:1  2.25 Ether 1.6:1   4.33 THF (0-rt) 1:1 12:1  7.58

ZnMe₂ (S:R) Solvent/T (° C.) U A^(Bz) THF (0-rt) NR Slow 2:1 DCM/TolueneVery 9:1 (−78-rt) slow

Dependence of Stereoselectivity on 2′-Substitution:

coordinating and more bulky 2′-OMe substituent gave better selectivitythan smaller, non-coordinating 2′-F or 2′-H.

b. Procedure for the Reaction of 5′-Oxo-Nucleosides withTrimethylaluminum.

5′-(S)—C-Methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-uridine3a and5′-(R)—C-Methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-uridine4a. A solution of crude aldehyde 2a, containing approximately 68% of thetitle compound (25.3 g, ≤68 mmol) in anhydrous THF (200 mL) was slowlyadded via cannula for approximately 15 minutes to a stirred and cooled(0° C.) mixture of AlMe₃ (2 M in heptane, 102 mL, 204 mmol) andanhydrous THF (200 mL) under argon atmosphere. The cooling bath wasremoved, the mixture was stirred at room temperature for 17 hours,cooled to 0° C. and the reaction was quenched by cautious addition of500 mL of 1:1 mixture of saturated aqueous solution of ammonium chlorideand 20% phosphoric acid followed by addition of 400 mL of ethylacetate.The organic phase was separated, washed twice with saturate brine, driedover anhydrous sodium sulfate, and the solvent was removed in vacuum toafford 25.6 g of crude residue. Flash column chromatography of theresidue on a 330 g CombiFlash silica gel column with gradient (50 to90%) of ethyl acetate containing 1% of triethylamine in hexanes afforded3a (17.5 g, 67%), and a mixture of 3a and 4a (1.25 g, 5%). The latterwas dissolved in 15 mL of hot ethyl acetate followed by slow addition of15 mL of hexanes. The mixture was allowed to cool to room temperature,stirred overnight, the precipitate was filtered, washed with ethylacetate-hexanes 1:2 mixture and dried to afford pure 4a (0.81 g, 65% oncrystallization, 3% on reaction). 3a: ¹H NMR (400 MHz, DMSO-d₆): δ 0.08(s, 6H); 0.87 (s, 9H); 1.14 (d, J=6.7 Hz, 3H); 3.33 (s, 3H); 3.68 (dd,J=1.8, 4.4 Hz, 1H); 3.76-3.84 (m, 2H); 4.27 (t, J=4.6 Hz, 1H, H2′); 5.17(d, J=4.4 Hz, 1H, OH); 5.65 (d, J=8.1 Hz, 1H); 5.83 (d, J=4.7 Hz, 1H,HP); 8.05 (d, J=8.1 Hz, 1H); 11.3 (s, 1H). ¹³C NMR (126 MHz, DMSO-d₆): δ−4.95; −4.82; 17.78; 20.05; 25.61; 57.56; 64.73; 70.57; 82.61; 85.83;87.96; 101.79; 140.19; 150.48; 163.08. HRMS m/z calc. for[C₁₇H₃₀N₂O₆Si+H]⁺: 387.1951; found: 387.1962. 4a: ¹H NMR (400 MHz,DMSO-d₆): δ 0.09 (s, 6H); 0.88 (s, 9H); 1.10 (d, J=6.6 Hz, 3H); 3.28 (s,3H); 3.64 (dd, J=2.2, 4.2 Hz, 1H, H4′); 3.77 (dt, J=4.6, 6.5 Hz, 1H,H5′); 3.86 (dd, J=4.8, 6.8 Hz, 1H, H2′); 4.40 (dd, J=2.2, 4.7 Hz, 1H,H3′); 5.16 (d, J=4.9 Hz, 1H, OH); 5.67 (dd, J=2.2, 8.1 Hz, 1H); 5.86 (d,J=6.8 Hz, 1H, H1′); 7.89 (d, J=8.2 Hz, 1H); 11.36 (d, J=1.8 Hz, 1H). ¹³CNMR (126 MHz, DMSO-d₆): δ 0.60; 0.69; 23.19; 25.19; 62.84; 71.50; 74.48;87.10; 90.47; 94.80; 107.69; 145.93; 156.11; 168.37. HRMS m/z calc. for[C₁₇H₃₀N₂O₆Si+H]⁺: 387.1951; found: 387.1960.

5′-(S)—C-Methyl-2′-deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-fluoro-uridine3b and5′-(R)—C-Methyl-2′-deoxy-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-fluoro-uridine4b were prepared analogously by addition of a solution of crude aldehyde2b containing approximately 55% of the title compound (1.80 g, ≤5 mmol)in anhydrous THF (10 mL) to a mixture of AlMe₃ (2 M in heptane, 8 mL, 16mmol) and anhydrous THF (10 mL) at 0° C. under argon atmosphere,followed by stirring at room temperature overnight. The crude residue(1.88 g) was chromatographed over a 80 g CombiFlash silica gel columnwith 50% of ethyl acetate containing 1% of triethylamine in hexanes toafford 3b (0.88 g, 47%), and 4b (0.17 g, 9%) along with smallintermediate mixed fraction. 3b: ¹H NMR (400 MHz, DMSO-d₆): δ 0.088 (s,3H); 0.094 (s, 3H); 0.86 (s, 9H); 1.19 (d, J=6.5 Hz, 3H); 3.72 (d, J=6.8Hz, 1H); 3.75-3.83 (m, 1H); 4.31 (ddd, J=4.4, 6.8, 18.3 Hz, 1H, H3′);5.06 (ddd, J=2.4, 4.4, 53.1 Hz, 1H, H2′); 5.20 (d, J=4.7 Hz, 1H, OH);5.63 (d, J=8.1 Hz, 1H); 5.91 (dd, J=2.3, 16.9 Hz, 1H, H1′); 7.99 (d,J=8.1 Hz, 1H); 11.4 (s, 1H). ¹³C NMR (126 MHz, acetone-d₆): δ −4.84;−4.53; 18.75; 20.70; 26.14; 66.02; 71.29; 71.42; 87.98; 88.50; 88.77;93.20; 94.71; 102.58; 141.37; 151.41; 163.74. ¹⁹F NMR (376 MHz,acetone-d₆): δ −207.60 (dt, J=16.6, 53.1 Hz, 1F). HRMS m/z calc. for[C₁₆H₂₇FN₂O₅Si+H]⁺: 375.1752; found: 375.1744. 4b: ¹H NMR (400 MHz,DMSO-d₆): δ 0.096 (s, 3H); 0.102 (s, 3H); 0.87 (s, 9H); 1.11 (d, J=6.7Hz, 3H); 3.74-3.78 (m, 1H); 3.84-3.94 (m, 1H); 4.43 (dt, J=4.8, 12.2 Hz,1H, H3′); 5.10 (dt, J=4.2, 52.8 Hz, 1H, H2′); 5.21 (d, J=4.7 Hz, 1H,OH); 5.65 (d, J=8.1 Hz, 1H); 5.94 (dd, J=4.0, 15.8 Hz, 1H, H1′); 7.89(d, J=8.1 Hz, 1H); 11.4 (s, 1H). ¹³C NMR (126 MHz, CD₃OD): δ −4.56;−4.10; 19.11; 19.89; 26.45; 67.67; 70.62; 70.74; 88.28; 88.55; 89.99;92.89; 94.40; 103.39; 142.71; 152.28; 165.67. ¹⁹F NMR (376 MHz,acetone-d₆): δ −208.28 (dt, J=14.3, 52.8 Hz, 1F). FIRMS m/z calc. for[C₁₆H₂₇FN₂O₅Si+H]⁺: 375.1752; found: 375.1760.

5′-(S)—C-Methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-thymidine 3c and5′-(R)—C-Methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-thymidine 4ewere prepared analogously by addition of a solution of crude aldehyde 2ccontaining approximately 60% of the title compound (1.21 g, ≤3.4 mmol)in anhydrous THF (10 mL) to a mixture of AlMe₃ (2 M in heptane, 6 mL, 12mmol) and anhydrous THF (10 mL) at 0° C. under argon atmosphere,followed by stirring at room temperature overnight. The crude residue(1.21 g) was chromatographed over 2 consecutive silica gel flash-columns(CombiFlash 80 g and 24 g) with gradient (80 to 100%) of ethyl ethercontaining 1% of triethylamine in hexanes. The fractions containingseparated epimers were pulled separately, and intermediate mixedfractions were combined and subjected to the second columnchromatography. Obtained 0.65 g (52%) of 3c and 0.11 g (9%) of 4c alongwith small intermediate mixed fraction. 3c: ¹H NMR (400 MHz, DMSO-d₆): δ0.07 (s, 6H); 0.86 (s, 9H); 1.13 (d, J=6.5 Hz, 3H); 1.76 (d, J=1.0 Hz,3H); 2.01 (ddd, J=3.0, 6.0, 13.2 Hz, 1H, H2′A); 2.13 (ddd, J=5.9, 7.7,13.4 Hz, 1H, H2 B); 3.58 (t, J=2.7 Hz, 1H, H4′); 3.74-3.83 (m, 1H, H5′);4.40 (quintet, J=2.8 Hz, 1H, H3′); 5.02 (d, J=4.6 Hz, 1H, OH); 6.15 (dd,J=6.0, 7.7 Hz, 1H, H1′); 7.84 (d, J=1.2 Hz, 1H); 11.27 (s, 1H). ¹³C NMR(126 MHz, ACN-d₃): δ −4.58; −4.39; 12.72; 18.63; 20.65; 26.20; 41.14;67.55; 73.94; 85.97; 97.74; 111.06; 137.89; 151.76; 165.10. HRMS m/zcalc. for [C₁₇H₃₀N₂O₅Si+Na]⁺: 393.1822; found: 393.1825. 4c: ¹H NMR (400MHz, DMSO-d₆): δ 0.081 (s, 3H); 0.084 (s, 3H); 0.87 (s, 9H); 1.10 (d,J=6.5 Hz, 3H); 1.76 (d, J=1.1 Hz, 3H); 1.95 (ddd, J=1.7, 5.5, 13.1 Hz,1H, H2′_(A)); 2.14 (ddd, J=5.4, 9.0, 13.2 Hz, 1H, H2′_(B)); 3.55 (dd,J=1.6, 4.7 Hz, 1H, H4′); 3.73 (dt, J=4.9, 6.4 Hz, 1H, H5′); 4.49 (dt,J=1.4, 5.3 Hz, 1H, H3′); 5.03 (d, J=5.0 Hz, 1H, OH); 6.15 (dd, J=5.5,8.9 Hz, 1H, H1′); 7.66 (d, J=1.2 Hz, 1H); 11.29 (s, 1H). ¹³C NMR (126MHz, ACN-d₃): δ −4.52; −4.25; 12.67; 18.55; 20.21; 26.19; 41.10; 68.26;72.46; 86.07; 92.35; 111.27; 137.75; 151.83; 165.09. HRMS m/z calc. for[C₁₇H₃₀N₂O₅Si+H]⁺: 371.2002; found: 371.1992.

N-Acetyl-5′-(S)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methylcytidine3d was prepared analogously by addition of a solution of crude aldehyde2d containing approximately 56% of the title compound (2.80 g, ≤6.8mmol) in anhydrous THF (20 mL) to a mixture of AlMe₃ (2 M in heptane, 12mL, 24 mmol) and anhydrous THF (20 mL) at 0° C. under argon atmosphere,followed by stirring at room temperature overnight. The crude residue(3.03 g) was chromatographed over 2 consecutive silica gel flash-columns(CombiFlash 80 g and 40 g) with gradient (70 to 100%) of ethyl acetatein hexanes to afford 1.09 g (37%) of less-polar (5)-epimer 3d along with0.60 g of more polar fraction containing mixture of 3d and 4d that wasnot further separated. 3d: ¹H NMR (400 MHz, DMSO-d₆): δ 0.05 (s, 6H);0.85 (s, 9H); 1.21 (d, J=6.5 Hz, 3H); 2.09 (s, 3H); 3.43 (s, 3H);3.70-3.76 (m, 2H); 3.77-3.85 (m, 1H); 4.21 (dd, J=4.8, 7.0 Hz, 1H); 5.19(d, J=4.4 Hz, 1H); 5.83 (d, J=2.0 Hz, 1H); 7.18 (d, J=7.5 Hz, 1H); 8.58(d, J=7.5 Hz, 1H); 10.90 (s, 1H).

Note:

N-Acetyl cytidines are not very stable on silica gel column in thepresence of triethylamine and tend to undergo disproportionation to formN-deprotected and N-diacetylated derivatives. Therefore, notriethylamine was used to separate the isomers 3d and 4d. However,addition of triethylamine is useful for better separation of the isomerson TLC.

N-Benzoyl-5′-(S)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-adenosine3e andN-benzoyl-5′-(R)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-adenosine4e were prepared analogously by addition of a solution of crude aldehyde2e containing approximately 50% of the title compound (16.9 g, ≤34 mmol)in anhydrous THF (100 mL) to a mixture of AlMe₃ (2 M in heptane, 51 mL,102 mmol) and anhydrous THF (100 mL) at 0° C. under argon atmosphere,followed by stirring at room temperature overnight. The crude residue(15.9 g) was chromatographed over a silica gel flash-column (CombiFlash220 g) with gradient (70 to 100%) of ethyl acetate in hexanes to affordpurified mixture of epimers (˜1:1): 8.52 g, 49%. The isomers werefurther separated by prep. C18 RP-HPLC using a Gilson PLC 2020purification system: 1 g of mixture was injected and eluted using anisocratic method with 25 mM triethylammonium bicarbonate and 65%acetonitrile. Appropriate fractions with HPLC purity of >95% were pooledand evaporated to dryness to afford 0.15 g of 3e (dr>97%), and 0.25 g of4e pure stereoisomer. 3e: ¹H NMR (400 MHz, DMSO-d₆): δ 0.124 (s, 3H);0.126 (s, 3H); 0.91 (s, 9H); 1.17 (d, J=6.4 Hz, 3H); 3.32 (s, 3H);3.81-3.90 (m, 2H); 4.40 (dd, J=4.9, 5.7 Hz, 1H); 4.54 (dd, J=3.2, 4.5Hz, 1H); 5.19 (d, J=5.7 Hz, 1H); 6.16 (d, J=5.7 Hz, 1H); 7.55 (t split,J=7.8 Hz, 2H); 7.64 (t split, J=7.4 Hz, 1H); 8.03 (d, J=1.4 Hz, 1H);8.05 (s, 1H); 8.76 (s, 1H); 8.80 (s, 1H); 11.23 (s, 1H). 4e: ¹H NMR (500MHz, DMSO-d₆): δ 0.146 (s, 3H); 0.147 (s, 3H); 0.93 (s, 9H); 1.10 (d,J=6.4 Hz, 3H); 3.25 (s, 3H); 3.75 (dd, J=1.1, 5.6 Hz, 1H); 3.89 (sextet,J=5.7 Hz, 1H); 4.60 (dd, J=4.5, 7.4 Hz, 1H); 4.63 (dd, J=1.2, 4.6 Hz,1H); 5.32 (d, J=4.7 Hz, 1H); 6.11 (d, J=7.4 Hz, 1H); 7.55 (t, J=8.0 Hz,2H); 7.64 (t split, J=7.5 Hz, 1H); 8.03 (d, J=1.4 Hz, 1H); 8.05 (d,J=0.9 Hz, 1H); 8.76 (s, 1H); 8.77 (s, 1H); 11.23 (s, 1H).

N-Isobutyryl-5′-(S)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-guanosine3f andN-Isobutyryl-5′-(R)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-guanosine4f were prepared analogously by addition of a solution of crude aldehyde2f containing approximately 53% of the title compound (1.63 g, ≤3.4mmol) in anhydrous DCM (10 mL) to a mixture of AlMe₃ (2 M in heptane, 10mL, 20 mmol) and anhydrous DCM (10 mL) at −78° C. under argonatmosphere, followed by slow (in bath) warming up to room temperatureovernight. The crude residue (1.59 g) was chromatographed over a silicagel flash-column (CombiFlash 40 g) with gradient (0 to 4%) of methanolin chloroform to afford 0.14 g of less polar (R)-isomer 4f, 0.36 g ofintermediate mixture of 3f and 4f and 0.25 g of more polar (5)-isomer3f. The intermediate fraction was separated on the second isocraticcolumn (CombiFlash 40 g) with 3% of methanol in chloroform to affordadditional 0.20 g of 4f and 0.16 g of 3f. Total yield of 3f: 0.34 g(23%) and 4f: 0.41 g (27%). 3f: ¹H NMR (400 MHz, DMSO-d₆): δ 0.10 (s,3H); 0.11 (s, 3H); 0.89 (s, 9H); 1.11 (d, J=6.8 Hz, 6H); 1.12 (d, J=6.4Hz, 3H); 2.77 (septet, J=6.8 Hz, 1H); 3.29 (s, 3H); 3.76 (t, J=2.6 Hz,1H); 3.79-3.87 (m, 1H); 4.20 (dd, J=4.8, 6.3 Hz, 1H); 4.44 (dd, J=2.6,4.6 Hz, 1H); 5.12 (d, J=4.6 Hz, 1H); 5.88 (d, J=6.3 Hz, 1H); 8.36 (s,1H); 11.61 (s, 1H); 12.10 (s, 1H). 4f: ¹H NMR (400 MHz, DMSO-d₆): δ 0.12(s, 3H); 0.13 (s, 3H); 0.90 (s, 9H); 1.08 (d, J=6.4 Hz, 3H); 1.11 (d,J=6.8 Hz, 6H); 2.76 (septet, J=6.8 Hz, 1H); 3.25 (s, 3H); 3.66 (d, J=5.6Hz, 1H); 3.76 (sextet, J=6.0 Hz, 1H); 4.36 (dd, J=4.6, 7.8 Hz, 1H); 4.54(d, J=4.5 Hz, 1H); 5.16 (d, J=5.1 Hz, 1H); 5.83 (d, J=7.8 Hz, 1H); 8.32(s, 1H); 11.60 (s, 1H); 12.10 (s, 1H).

c. Procedure for the Stereoselective Reaction of Purine5′-Oxo-Nucleosides with Dimethylzinc.

N-Benzoyl-5′-(S)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-adenosine3e. A solution of crude aldehyde 2e, containing approximately 55% of thetitle compound (1.69 g, ≤3.4 mmol) in anhydrous DCM (10 mL) was slowlyadded dropwise for approximately 20 minutes to a stirred and cooled(−78° C.) mixture of ZnMe₂ (2 M in toluene, 6 mL, 12 mmol) and anhydrousDCM (10 mL) under argon atmosphere. The solution was allowed to slowlywarm up (in bath) to room temperature overnight, cooled to 0° C., andquenched by cautious addition of 10% phosphoric acid. The organic phasewas separated, washed with 5% brine, and dried over anhydrous sodiumsulfate. The solvent was removed in vacuum and the crude residue (1.57g) was purified by flash chromatography on a 40 g CombiFlash silica gelcolumn with gradient (70 to 100%) of ethyl acetate in hexanes to afford3e (0.96 g, 55%) in ˜90% diastereomeric purity. The compound wasdissolved in 5 mL of diethyl ether, and hexane (4 mL) was slowly addedwith stirring that triggered crystallization. The mixture was stirred atroom temperature overnight, filtered and the solids were washed twicewith ether-hexanes 1:2 mixture to afford 0.69 g (73% on crystallization)of 3e of ˜97% diastereomeric purity.

N-Isobutyryl-5′-(S)—C-methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-O-methyl-guanosine3f was prepared analogously by addition of a solution of crude aldehyde2f containing approximately 53% of the title compound (1.63 g, ≤3.4mmol) in anhydrous DCM (10 mL) to a mixture of ZnMe₂ (2 M in toluene,8.5 mL, 17 mmol) and anhydrous DCM (10 mL) at −78° C. under argonatmosphere, followed by slow (in bath) warming up to room temperatureovernight. The crude residue (1.56 g) containing ˜3:1 ratio of 3f to 4fwas chromatographed over a silica gel flash-column (CombiFlash 40 g)with 2% of methanol in chloroform to afford 0.13 g of mixture of 4f and3f followed by pure 3f 0.48 g (32%).

d. Stereospecific Interconversion of 5′-Alkyl-Epimers

5′-(S)—C-methyl-5′-O-mesyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-uridine5a. Methanesulfonyl chloride (5.5 mL, 72 mmol) was added dropwise over aperiod of ˜5 minutes so a cooled (0° C.) and stirred solution of(S)-epimer (3a) (8.32 g, 21.6 mmol) and anhydrous pyridine (5.8 mL, 72mmol) in anhydrous DCM (80 mL) under argon atmosphere. The cooling bathwas removed, the mixture was stirred at room temperature for 48 hours,cooled to 0° C., and quenched by careful addition of saturated solutionof sodium bicarbonate (200 mL). The cooling bath was removed, themixture was stirred vigorously at room temperature for 1 hour, theorganic phase was separated, washed consecutively with 10% phosphoricacid, twice with 5% brine, and dried over anhydrous sodium sulfate. Thesolvent was removed in vacuum and the residue was dried under highvacuum to afford essentially pure 5a (9.79 g, 98%) as an orange foam.5a: ¹H NMR (400 MHz, DMSO-d₆): δ 0.09 (s, 3H); 0.10 (s, 3H); 0.87 (s,9H); 1.42 (d, J=6.5 Hz, 3H); 3.20 (s, 3H); 3.33 (s, 3H); 3.86 (t, J=4.9Hz, 1H); 3.89 (t, J=4.9 Hz, 1H); 4.29 (t, J=5.1 Hz, 1H); 4.91 (dt,J=6.4, 11.3 Hz, 1H); 5.68 (dd, J=2.2, 8.1 Hz, 1H); 5.86 (d, J=4.7 Hz,1H); 7.65 (d, J=8.2 Hz, 1H); 11.42 (s, 1H).

6,9-Epoxy-2H,6H-pyrimido[2,1-b][1,3]oxazacin-2-one-7,8,10-trihydro-9-(R)-methyl-8-O-[(1,1-dimethylethyl)dimethylsilyl]-7-methoxy-[6R-(6α,7α,8α,9α)]6a. A solution of mesylate 5a (2.33 g, 5 mmol and DBU (1.5 mL, 10 mmol)in anhydrous DMSO (10 mL) was stirred at 60° C. under argon atmospherefor 27 hours, cooled to 0° C., and ethyl acetate (40 mL) followed by 5%aqueous NaCl (80 mL) were added. The organic phase was separated, washedwith a 1:1 mixture of 5% NaCl and 10% aqueous phosphoric acid, followedby 5% NaCl, saturated sodium bicarbonate, and saturated NaCl. Afterdrying over anhydrous sodium sulfate, the solvent was removed in vacuumto afford crude anhydrous product 6a (1.57 g), that was refluxed with 30mL of diethyl ether for 45 minutes, cooled to room temperature, stirredfor 2 hours, the white precipitate was filtered, washed twice withdiethyl ether and dried to afford 0.88 g (48%) of pure 6a. 6a: ¹H NMR(400 MHz, DMSO-d₆): δ 0.06 (s, 3H); 0.7 (s, 3H); 0.84 (s, 9H); 1.36 (d,J=6.7 Hz, 3H); 3.29 (s, 3H); 4.13 (dd, J=0.8, 6.0 Hz, 1H); 4.27 (s, 1H);4.32 (q, J=6.8 Hz, 1H); 4.62 (d, J=5.9 Hz, 1H); 5.78 (s, 1H); 5.89 (d,J=7.4 Hz, 1H); 7.95 (d, J=7.4 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆): δ−5.22; −4.81; 16.79; 17.91; 25.59; 57.93; 71.42; 81.77; 86.33; 91.16;96.24; 109.09; 142.58; 156.29; 170.67.

5′-(R)—C-Methyl-3′-O-[(1,1-dimethylethyl)dimethylsilyl]-2′-O-methyl-uridine4a (from 5a), A solution of mesylate 5a (3.72 g, 8 mmol), DBU (2.4 mL,16 mmol), and water (10 mL) in THF (50 mL) was refluxed under argonatmosphere for 67 hours. The solvent was removed in vacuum, the residuewas partitioned between ethyl acetate (120 mL) and a mixture of 80 mL of5% NaCl and 30 mL of 10% phosphoric acid, the organic phase wasseparated, washed twice with 5% NaCl followed by saturated NaCl. Afterdrying over anhydrous sodium sulfate, the crude residue (3.04 g) wasrefluxed with a mixture of 30 mL of diethyl ether and 15 mL of hexanesfor 1 hour, cooled to room temperature, stirred overnight, the whiteprecipitate was filtered, and washed twice with ether-hexanes 1:1mixture to afford 2.32 g (75%) of 4a.

The process described in this example can be used for the synthesis ofvarious 5′-alkyl nucleosides for any therapeutic applications (e.g.,antiviral, and antitumor applications), including oligonucleotide andsmall molecules.

Example 9: Steric Blocking of Phosphodiester (PO), Phosphorothioate (PS)and Phosphorodithioate (PS2) by Introducing 4′ and 5′-ModifiedNucleotide to the 3′-End of PO, PS, or PS2 Linkage

The inventors found that introducing a 4′-modified and/or 5′-modifiednucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate(PS), and/or phosphorodithioate (PS2) linkage of a dinucleotide at anyposition of single stranded or double stranded oligonucleotide can exertsteric effect to the internucleotide linkage and, hence, protecting orstabilizing it against nucleases.

In this example, in vitro gene silencing activity of F7 siRNA containing4′-O-methylated or 5′-methylated nucleotides at selected position wereevaluated, and the results are shown in Table 11.

TABLE 11 in vitro gene silencing activity of F7 siRNAcontaining 4′-O-methylated or 5′-methylatednucleotides at selected position (Table 11 discloses SEQ ID NOS 233-274,respectively, in order of columns) Duplex 10 nM 0.1 nM ID SenseAntisense vg D vg D D-60347 CfsasGfgAfuCfaUfCfUfc gsUfsaAfgAfcUfuGfaga3.2 .9 1.4 .2 AfaGfuCfuUfaCfL96 UfgAfuCfcUfgsgsc D-63931CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .7 .7 1.2 .7AfaGfuCfUUfaAfL96 UfgAfuCfcUfgsgsc D-69122 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .9 .7 1.7 .7 AfaGfuCfU(Ufm)aAfL96 UfgAfuCfcUfgsgscD-69123 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .4 .9 0.6 .5AfaGfuCfU(UfmR)aAfL96 UfgAfuCfcUfgsgsc D-69124 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .4 .1 3.9 .0 AfaGfuCfU(u5m)aAfL96 UfgAfuCfcUfgsgscD-69125 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .5 .6 5.1 .0AfaGfuCfU(u5mR)aAfL96 UfgAfuCfcUfgsgsc D-69126 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .9 .4 0.7 .9 AfaGfuCfU(T5m)aAfL96 UfgAfuCfcUfgsgscD-69127 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .5 .5 4.0 .5AfaGfuCfU(T5mR)aAfL96 UfgAfuCfcUfgsgsc D-69128 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .6 .3 0.4 .1 AfaGfuCfU(Ufo)aAfL96 UfgAfuCfcUfgsgscD-69129 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .3 .2 0.9 .3AfaGfuCfU(dTo)aAfL96 UfgAfuCfcUfgsgsc D-69130 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .6 .7 1.6 .5 AfaGfuCfUuaAfL96 UfgAfuCfcUfgsgscD-69131 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .7 .3 4.8 .1AfaGfuCfUdTaAfL96 UfgAfuCfcUfgsgsc D-63934 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .2 .7 9.2 .4 AfaGfuCfuUfaAfL96 UfgAfUCfcUfgsgscD-69132 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .9 .2 7.7 .1AfaGfuCfuUfaAfL96 UfgAfU(Cfm)cUfgsgsc D-69133 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .5 .9 8.2 .1 AfaGfuCfuUfaAfL96UfgAfU(CfmR)cUfgsgsc D-69134 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga .4 .7 3.1 .7 AfaGfuCfuUfaAfL96 UfgAfU(c5m)cUfgsgsc D-69135CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .5 .8 7.8 .2AfaGfuCfuUfaAfL96 UfgAfU(c5mR)cUfgsgsc D-69136 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .5 .8 5.3 .1 AfaGfuCfuUfaAfL96 UfgAfU(Cfo)cUfgsgscD-69137 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .1 .4 8.6 .9AfaGfuCfuUfaAfL96 UfgAfU(dCo)cUfgsgsc D-69138 CfsasGfgAfuCfaUfCfUfcusUfsaAfgAfcUfuGfaga  .1 .8 8.9 .9 AfaGfuCfuUfaAfL96 UfgAfUccUfgsgscD-69139 CfsasGfgAfuCfaUfCfUfc usUfsaAfgAfcUfuGfaga  .1 .6 9.6 .1AfaGfuCfuUfaAfL96 UfgAfUdTcUfgsgsc

  2′-fluoro-5′-(S)-methyl-uridine-3′- phosphate (Ufm)

  2′-fluoro-5′-(S)-methyl-uridine-3′- phosphorothioate (Ufms)

  2′-fluoro-5′-(R)-methyl-uridine-3′- phosphorothioate (UfmRs)

  2′-fluoro-5′-(R)-methyl-uridine-3′- phosphate (UfmR)

  2′-O-methyl-5′-(R)-methyl-uridine- 3′-phosphate (u5mR)

  2′-O-methyl-5′-(S)-methyl-uridine-3′- phosphate (u5m)

  2′-O-methyl-5′-(S)-methyl-cytidine- 3′-phosphate (c5m)

  2′-O-methyl-5′-(R)-methyl-cytidine-3′- phosphate (c5mR)

  5′-(S)-methyl-deoxythymidine-3′- phosphate (T5m)

  5′-(R)-methyl-deoxythymidine-3′- phosphate (T5mR)

  2′-fluoro-5′-(S)-methyl-cytosine-3′- phosphate (Cfm)

  2′-fluoro-5′-(S)-methyl-cytosine-3′- phosphorothioate (Cfms)

  2′-fluoro-5′-(R)-methyl-cytosine-3′- phosphate (CfmR)

  2′-fluoro-5′-(R)-methyl-cytosine-3′- phosphorothioate (CfmRs)

  2′-fluoro-4′-methoxyuridine-3′- phosphate ((Ufo)

  2′-fluoro-4′-methoxyuridine-3′- phosphorothioate (Ufos)

  4′-methoxy-2′-deoxythymidine-3′- phosphate (dTo)

  4′-methoxy-2′-deoxythymidine-3′- phosphorothioate (dTos)

  2′-fluoro-4′-methoxycytidine-3′- phosphate (Cfo)

  2′-fluoro-4′-methoxycytidine-3′- phosphorothioate (Cfos)

  4′-methoxy-2′-deoxycytidine-3′- phosphate (dCo)

  4′-methoxy-2′-deoxycytidine-3′- phosphorothioate (dCos)

Example 10: Chirality-Dependent Activity of Glycol Nucleic Acid (GNA) insiRNA Duplexes

Chemical modifications of siRNA duplexes are necessary to stabilizethese molecules against nuclease degradation, to facilitate their uptakeinto cells, and to affect formation of active RISC as well asRNAi-mediated target silencing. Thermally destabilizing modificationsincorporated at certain positions of the siRNA duplex can lead to anincrease in potency by improving strand bias and/or sense stranddissociation during RISC loading.

In this example, a three-carbon, acyclic nucleic acid analog, GlycolNucleic Acid (GNA) was evaluated within the context of siRNA duplexes.GNA-containing siRNA duplexes were synthesized. (S)-GNA oligomers formedhomo-duplexes with structural similarities to a typical RNA A-formduplex and crosspair with RNA, but not DNA, within A/T-rich sequences.The thermal stabilities and nuclease resistance of siRNA duplexescontaining (S)- or (R)-GNA were investigated. Structural studies usingx-ray crystallography provided further insight into the effect of theseGNA substituents within RNA duplexes. Chirality-dependent gene silencingactivity of GNA-containing siRNA duplexes was examined in biologicalstudies.

The table below discloses SEQ ID NOS 275-366, respectively, in order ofcolumns.

Duplex ID Sense (5′ to 3′) Antisense (5′ to 3′) Design AD-57727.66AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA Parent GfcUfcUfaUfaAfL96fcAfcUfgUfususu AD-68368.2 AfsasCfaGfuGfuUfCfUfu (Tgns)UfsaUfaGfaGfcAfAS: gN @ N1 GfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu AD-68369.2AfsasCfaGfuGfuUfCfUfu P(Tgns)UfsaUfaGfaGfcA AS: 5′-p, GfcUfcUfaUfaAfL96fagaAfcAfcUfgUfususu gN @ N2 AD-62896.4 AfsasCfaGfuGfuUfCfUfuus(Tgns)aUfaGfaGfcAfa AS: gN @ N2 GfcUfcUfaUfaAfL96 gaAfcAfcUfgUfususuAD-68370.2 AfsasCfaGfuGfuUfCfUfu usUfs(Agn)UfaGfaGfcAf AS: gN @ N3GfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu AD-68371.2 AfsasCfaGfuGfuUfCfUfuusUfsa(Tgn)aGfaGfcAfa AS: gN @ N4 GfcUfcUfaUfaAfL96 gaAfcAfcUfgUfususuAD-68372.2 AfsasCfaGfuGfuUfCfUfu usUfsaUf(Agn)GfaGfcAf AS: gN @ N5GfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu AD-68373.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfa(Ggn)aGfcAfa AS: gN @ N6 GfcUfcUfaUfaAfL96 gaAfcAfcUfgUfususuAD-68374.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGf(Agn)GfcAf AS: gN @ N7GfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu AD-68375.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfa(Ggn)cAfa AS: gN @ N8 GfcUfcUfaUfaAfL96 gaAfcAfcUfgUfususuAD-68376.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGf(Cgn)Af AS: gN @ N9GfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu AD-68377.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfc(Agn)a AS: gN @ N10 GfcUfcUfaUfaAfL96 gaAfcAfcUfgUfususuAD-68378.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAf AS: gN @ N11GfcUfcUfaUfaAfL96 (Agn)gaAfcAfcUfgUfusu su AD-68379.2AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfa AS: gN @ N12 GfcUfcUfaUfaAfL96(Ggn)aAfcAfcUfgUfusus u AD-68380.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfag AS: gN @ N13 GfcUfcUfaUfaAfL96 (Agn)AfcAfcUfgUfususuAD-68381.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfaga AS: gN @ N14GfcUfcUfaUfaAfL96 (Agn)cAfcUfgUfususu AD-68382.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA AS: gN @ N15 GfcUfcUfaUfaAfL96 f(Cgn)AfcUfgUfususuAD-68383.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA AS: gN @ N16GfcUfcUfaUfaAfL96 fc(Agn)cUfgUfususu AD-68384.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA AS: gN @ N17 GfcUfcUfaUfaAfL96 fcAf(Cgn)UfgUfususuAD-68385.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA AS: gN @ N18GfcUfcUfaUfaAfL96 fcAfc(Tgn)gUfususu AD-68386.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA AS: gN @ N19 GfcUfcUfaUfaAfL96 fcAfcUf(Ggn)UfususuAD-68387.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA AS: gN @ N20GfcUfcUfaUfaAfL96 fcAfcUfg(Tgn)ususu AD-68388.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA AS: gN @ N21 GfcUfcUfaUfaAfL96 fcAfcUfgUf(Tgns)usuAD-68389.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA AS: gN @ N22GfcUfcUfaUfaAfL96 fcAfcUfgUfus(Tgns)u AD-68390.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA AS: gN @ N23 GfcUfcUfaUfaAfL96 fcAfcUfgUfusus(Tgn)AD-68391.2 (Agns)asCfaGfuGfuUfCf usUfsaUfaGfaGfcAfagaA S: gN @ N1UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68392.2 Afs(Agns)CfaGfuGfuUfCusUfsaUfaGfaGfcAfagaA S: gN @ N2 fUfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususuAD-68393.2 Afsas(Cgn)aGfuGfuUfCf usUfsaUfaGfaGfcAfagaA S: gN @ N3UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68394.2 AfsasCf(Agn)GfuGfuUfCusUfsaUfaGfaGfcAfagaA S: gN @ N4 fUfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususuAD-68395.2 AfsasCfa(Ggn)uGfuUfCf usUfsaUfaGfaGfcAfagaA S: gN @ N5UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68396.2 AfsasCfaGf(Tgn)GfuUfCusUfsaUfaGfaGfcAfagaA S: gN @ N6 fUfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususuAD-68397.2 AfsasCfaGfu(Ggn)uUfCf usUfsaUfaGfaGfcAfagaA S: gN @ N7UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68398.2 AfsasCfaGfuGf(Tgn)UfCusUfsaUfaGfaGfcAfagaA S: gN @ N8 fUfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususuAD-68399.2 AfsasCfaGfuGfu(Tgn)Cf usUfsaUfaGfaGfcAfagaA S: gN @ N9UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68400.2 AfsasCfaGfuGfuUf(Cgn)usUfsaUfaGfaGfcAfagaA S: gN @ N10 UfuGfcUfcUfaUfaAfL96 fcAfcUfgUfususuAD-62900.1 AfsasCfaGfuGfuUfCf usUfsaUfaGfaGfcAfagaA S: gN @ N11(Tgn)uGfcUfcUfaUfaAf fcAfcUfgUfususu L96 AD-68401.2 AfsasCfaGfuGfuUfCfUfusUfsaUfaGfaGfcAfagaA S: gN @ N12 (Tgn)GfcUfcUfaUfaAf fcAfcUfgUfususuL96 AD-68402.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA S: gN @ N13(Ggn)cUfcUfaUfaAfL96 fcAfcUfgUfususu AD-68403.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA S: gN @ N14 Gf(Cgn)UfcUfaUfaAfL96 fcAfcUfgUfususuAD-68404.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA S: gN @ N15Gfc(Tgn)cUfaUfaAfL96 fcAfcUfgUfususu AD-68405.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA S: gN @ N16 GfcUf(Cgn)UfaUfaAfL96 fcAfcUfgUfususuAD-68406.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA S: gN @ N17GfcUfc(Tgn)aUfaAfL96 fcAfcUfgUfususu AD-68407.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA S: gN @ N18 GfcUfcUf(Agn)UfaAfL96 fcAfcUfgUfususuAD-68408.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA S: gN @ N19GfcUfcUfa(Tgn)aAfL96 fcAfcUfgUfususu AD-68409.2 AfsasCfaGfuGfuUfCfUfuusUfsaUfaGfaGfcAfagaA S: gN @ N20 GfcUfcUfaUf(Agn)AfL96 fcAfcUfgUfususuAD-68410.2 AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfagaA S: gN @ N21GfcUfcUfaUfa(Agn)L96 fcAfcUfgUfususu

The table below discloses SEQ ID NOS 367-456, respectively, in order ofcolumns.

Duplex ID Sense (5′ to 3′) Antisense (5′ to 3′) Design AD-57727.66AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGfcAfaga Parent GfcUfcUfaUfaAfL96AfcAfcUfgUfususu AD-69078.1 AfsasCfaGfuGfuUfCfUfu (Tgns)UfsaUfaGfaGfcAAS: gN @ N1; GfcUfcUfaUfa(Agn)L96 fagaAfcAfcUfgUfususu S: gN @ N21AD-69079.1 AfsasCfaGfuGfuUfCfUfu us(Tgns)aUfaGfaGfcAf AS: gN @ N2;GfcUfcUfaUf(Agn)AfL96 agaAfcAfcUfgUfususu S: gN @ N20 AD-69080.1AfsasCfaGfuGfuUfCfUfu usUfs(Agn)UfaGfaGfcA AS: gN @ N3;GfcUfcUfa(Tgn)aAfL96 fagaAfcAfcUfgUfususu S: gN @ N19 AD-69081.1AfsasCfaGfuGfuUfCfUfu usUfsa(Tgn)aGfaGfcAf AS: gN @ N4;GfcUfcUf(Agn)UfaAfL96 agaAfcAfcUfgUfususu S: gN @ N18 AD-69082.1AfsasCfaGfuGfuUfCfUfu usUfsaUf(Agn)GfaGfcA AS: gN @ N5;GfcUfc(Tgn)aUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N17 AD-69083.1AfsasCfaGfuGfuUfCfUfu usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;GfcUf(Cgn)UfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N16 AD-69084.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGf(Agn)GfcA AS: gN @ N7;Gfc(Tgn)cUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N15 AD-69085.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfa(Ggn)cAf AS: gN @ N8;Gf(Cgn)UfcUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N14 AD-69086.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfaGf(Cgn)A AS: gN @ N9;(Ggn)cUfcUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N13 AD-69087.1AfsasCfaGfuGfuUfCfUf usUfsaUfaGfaGfc(Agn) AS: gN @ N10;(Tgn)GfcUfcUfaUfaAf agaAfcAfcUfgUfususu S: gN @ N12 L96 AD-69088.1AfsasCfaGfuGfuUfCf usUfsaUfaGfaGfcAf AS: gN @ N11; (Tgn)uGfcUfcUfaUfaAf(Agn)gaAfcAfcUfgUfus S: gN @ N11 L96 usu AD-69089.1AfsasCfaGfuGfuUf(Cgn) usUfsaUfaGfaGfcAfa AS: gN @ N12;UfuGfcUfcUfaUfaAfL96 (Ggn)aAfcAfcUfgUfusu S: gN @ N10 su AD-69090.1AfsasCfaGfuGfu(Tgn)Cf usUfsaUfaGfaGfcAfag AS: gN @ N13;UfuGfcUfcUfaUfaAfL96 (Agn)AfcAfcUfgUfusus S: gN @ N9 u AD-69091.1AfsasCfaGfuGf(Tgn)UfC usUfsaUfaGfaGfcAfaga AS: gN @ N14;fUfuGfcUfcUfaUfaAfL96 (Agn)cAfcUfgUfususu S: gN @ N8 AD-69092.1AfsasCfaGfu(Ggn)uUfCf usUfsaUfaGfaGfcAfaga AS: gN @ N15;UfuGfcUfcUfaUfaAfL96 Af(Cgn)AfcUfgUfususu S: gN @ N7 AD-69093.1AfsasCfaGf(Tgn)GfuUfC usUfsaUfaGfaGfcAfaga AS: gN @ N16;fUfuGfcUfcUfaUfaAfL96 Afc(Agn)cUfgUfususu S: gN @ N6 AD-69094.1AfsasCfa(Ggn)uGfuUfCf usUfsaUfaGfaGfcAfaga AS: gN @ N17;UfuGfcUfcUfaUfaAfL96 AfcAf(Cgn)UfgUfususu S: gN @ N5 AD-69095.1AfsasCf(Agn)GfuGfuUfC usUfsaUfaGfaGfcAfaga AS: gN @ N18;fUfuGfcUfcUfaUfaAfL96 AfcAfc(Tgn)gUfususu S: gN @ N4 AD-69096.1Afsas(Cgn)aGfuGfuUfCf usUfsaUfaGfaGfcAfaga AS: gN @ N19;UfuGfcUfcUfaUfaAfL96 AfcAfcUf(Ggn)Ufususu S: gN @ N3 AD-69097.1Afs(Agns)CfaGfuGfuUfC usUfsaUfaGfaGfcAfaga AS: gN @ N20;fUfuGfcUfcUfaUfaAfL96 AfcAfcUfg(Tgn)ususu S: gN @ N2 AD-69098.1(Agns)asCfaGfuGfuUfCf usUfsaUfaGfaGfcAfaga AS: gN @ N21;UfuGfcUfcUfaUfaAfL96 AfcAfcUfgUf(Tgns)usu S: gN @ N1 AD-69099.1AfsasCfaGfuGfuUfCfUfu usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;Gfc(Tgn)cUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N15 AD-69100.1AfsasCfaGfuGfuUfCfUfu usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;GfcUfc(Tgn)aUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N17 AD-69101.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGf(Agn)GfcA AS: gN @ N7;GfcUf(Cgn)UfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N16 AD-69102.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGf(Agn)GfcA AS: gN @ N7;GfcUfc(Tgn)aUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N17 AD-69103.1AfsasCfaGfuGfuUfCfUfu usUfs(Agn)UfaGfaGfcA AS: gN @ N3;Gfc(Tgn)cUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N15 AD-69104.1AfsasCfaGfuGfuUfCfUfu usUfs(Agn)UfaGfaGfcA AS: gN @ N3;GfcUf(Cgn)UfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N16 AD-69105.1AfsasCfaGfuGfuUfCfUfu usUfs(Agn)UfaGfaGfcA AS: gN @ N3;GfcUfc(Tgn)aUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N17 AD-69106.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfa(Ggn)cAf AS: gN @ N8;Gfc(Tgn)cUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N15 AD-69107.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfa(Ggn)cAf AS: gN @ N8;GfcUf(Cgn)UfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N16 AD-69108.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGfa(Ggn)cAf AS: gN @ N8;GfcUfc(Tgn)aUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N17 AD-69109.1Afs(Agns)CfaGfuGfuUfC usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;fUfuGfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N2 AD-69110.1Afs(Agns)CfaGfuGfuUfC usUfsaUfaGf(Agn)GfcA AS: gN @ N7;fUfuGfcUfcUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N2 AD-69111.1AfsasCfaGf(Tgn)GfuUfC usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;fUfuGfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N6 AD-69112.1AfsasCfaGf(Tgn)GfuUfC usUfsaUfaGf(Agn)GfcA AS: gN @ N7;fUfuGfcUfcUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N6 AD-69113.1AfsasCfaGfu(Ggn)uUfCf usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;UfuGfcUfcUfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N7 AD-69114.1AfsasCfaGfu(Ggn)uUfCf usUfsaUfaGf(Agn)GfcA AS: gN @ N7;UfuGfcUfcUfaUfaAfL96 fagaAfcAfcUfgUfususu S: gN @ N7 AD-69115.1AfsasCfaGfuGfuUfCfUfu usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;GfcUfcUfaUfa(Agn)L96 agaAfcAfcUfgUfususu S: gN @ N21 AD-69116.1AfsasCfaGfuGfuUfCfUfu usUfsaUfaGf(Agn)GfcA AS: gN @ N7;GfcUfcUfaUfa(Agn)L96 fagaAfcAfcUfgUfususu S: gN @ N21 AD-69117.1AfsasCfaGfuGfuUfCf us(Tgns)aUfaGfaGfcAf AS: gN @ N2;(Tgn)uGfcUfcUfaUfaAf agaAfcAfcUfgUfususu S: gN @ N11 L96 AD-69118.1AfsasCfaGfuGfuUfCfUf us(Tgns)aUfaGfaGfcAf AS: gN @ N2;(Tgn)GfcUfcUfaUfaAf agaAfcAfcUfgUfususu S: gN @ N12 L96 AD-69119.1AfsasCfaGfuGfuUfCf usUfsaUfa(Ggn)aGfcAf AS: gN @ N6;(Tgn)uGfcUfcUfaUfaAf agaAfcAfcUfgUfususu S: gN @ N11 L96 AD-69120.1AfsasCfaGfuGfuUfCf usUfsaUfaGf(Agn)GfcA AS: gN @ N7;(Tgn)uGfcUfcUfaUfaAf fagaAfcAfcUfgUfususu S: gN @ N11 L96 AD-69121.1AfsasCfaGfuGfuUfCfUfu us(Tgns)aUfaGfaGfcAf AS: gN @ N2;GfcUf(Cgn)UfaUfaAfL96 agaAfcAfcUfgUfususu S: gN @ N16

The results are shown in FIGS. 27-30.

FIG. 27 is a graph showing the in vitro knockdown of TTR using siRNAmodified with a single (S)-GNA nucleotide. Levels of TTR mRNA weremeasured after incubation with 10 nM siRNA in primary mouse hepatocytesfor 24 hours. TTR mRNA was assessed using RT-qPCR and normalized to PBStreated cells. All data points were the average of four measurements.FIG. 27 shows the influence of single (S)-GNA nucleotide incorporationon the in vitro siRNA activity.

FIG. 28A is a graph showing the in vitro knockdown of TTR using siRNAmodified with a single (S)-GNA base pair. Levels of TTR mRNA weremeasured after incubation with 10 nM siRNA in primary mouse hepatocytesfor 24 hours. TTR mRNA was assessed using RT-qPCR and normalized to PBStreated cells. All data points were the average of four measurements.FIG. 28B shows mix and match duplexes where sense and antisense strandscontaining single (S)-GNA nucleotides were paired as GNA:RNA hetero-basepairs. FIG. 28 shows the influence of single (S)-GNA base pairincorporation on in vitro siRNA activity

FIG. 29 is a graph showing the in vivo levels of TTR in mouse serum.Animals received a single dose of 2.5 mg/kg siRNA. At the indicated timepre- or post-dosing, animals were bled and serum samples were measuredusing a sandwich ELISA assay utilizing a HRP-conjugate antibody and3,3′,5,5′-tetramethylbenzidine for readout at 450 nm. All samples weremeasured in duplicate and each data point is the average of the mice ineach cohort (n=3). FIG. 29 illustrates the effect of in vivo genesilencing in mice using GNA-modified siRNA duplexes on serum TTR levels.

FIG. 30 is a graph showing the in vivo quantification of TTR mRNAlevels. Animals received a single dose of 2.5 mg/kg siRNA. At theindicated time post-dosing, RNA extraction was performed on whole-liverhomogenate. TTR mRNA was assessed as above by RT-qPCR, using the ΔΔCtmethod with GAPDH as the control transcript, and normalized toPBS-treated animals. FIG. 30 illustrates the effect of in vivo genesilencing in mice using GNA-modified siRNA duplexes on liver mRNAlevels.

The results shown in the above figures demonstrate that GNAincorporation resulted in a position-dependent thermal destabilizationof the resulting duplex. The extent of destabilization was nucleotidedependent; whereas substitution for an A or U nucleotide resulted in amuch smaller ATM compared to GNA substitution for G or C nucleotides.The incorporation of single GNA nucleotides into the seed region ofsiRNA duplexes resulted in similar levels of knockdown of TTR mRNA invitro. In addition, siRNA containing GNA base-pairs within the seedregion, as well as mix and match duplexes, demonstrated higher levels ofknockdown in vitro than the corresponding parent siRNA.

In vivo gene silencing correlated well with in vitro results forduplexes containing a single GNA substitution. Dual substitution of GNAresulted in a loss of in vivo silencing activity when compared to thesingle-substituted siRNAs.

All of the U.S. patents, U.S. patent application publications, foreignpatents, foreign patent applications and non-patent publicationsreferred to in this specification are incorporated herein by reference,in their entirety. Aspects of the embodiments can be modified, ifnecessary to employ concepts of the various patents, applications andpublications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

We claim:
 1. A double-stranded RNA (dsRNA) agent capable of inhibitingthe expression of a target gene, comprising a sense strand sequence andan antisense strand sequence complementary to at least one portion of amRNA of the target gene, each of the sense and antisense strands having19-25 nucleotides in length, wherein the sense strand is represented byformula (Is):

wherein: B1, B2, and B3 each independently represent a nucleotidecontaining a modification selected from the group consisting of2′-Oalkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA,and BNA/LNA; C1 is a thermally destabilizing nucleotide, selected fromthe group consisting of i) a nucleotide that forms a mismatch pair withthe opposing nucleotide in the antisense strand, ii) a nucleotide havingan abasic modification, and iii) a nucleotide having a sugarmodification, and placed at a site opposite to the seed region(positions 2-8) of the antisense strand; T1 represents a nucleotidecomprising a 2′-F modification; n¹ or n³ is independently 4 to 15nucleotides in length; n⁵ is 1-6 nucleotide(s) in length; n² is 3; n⁴ is0-3 nucleotide(s) in length; and wherein the sense strand has 2′-Fmodifications, and wherein the 2′-F modifications on the sense strandconsist of four, and only four, 2′-F modifications, wherein the four2′-F modifications are at positions 7 and 9-11 from the 5′-end ofthesense strand.
 2. The dsRNA agent of claim 1, wherein n⁴ is
 1. 3. ThedsRNA agent of claim 2, wherein C1 is at position 14-17 of the 5′-end ofthe sense strand, when the sense strand is 19-22 nucleotides in length,and n⁴ is
 1. 4. The dsRNA agent of claim 1, wherein C1 has thermallydestabilizing modification selected from the group consisting of abasicmodification selected from the group consisting of:

and sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar.
 5. The dsRNA agent ofclaim 1, wherein B1, B2, and B3 each contain 2′-OMe modifications. 6.The dsRNA agent of claim 1, wherein the sense strand comprises one blockof two phosphorothioate or methylphosphonate internucleotide linkages.7. The dsRNA agent of claim 1, wherein the dsRNA agent has a 3′ and/or5′ overhang(s) of 1-10 nucleotides in length.
 8. The dsRNA agent ofclaim 1, further comprising at least one ASGPR ligand attached to the 3′end of the sense strand.
 9. The dsRNA agent of claim 8, wherein theASGPR ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.
 10. The dsRNA agent of claim 9,wherein the ASGPR ligand is:


11. The dsRNA agent of claim 1, wherein formula (Is) further comprises a2′-deoxythymidine linked via a phosphorodithioate (PS₂) linkage or a5′-vinyl phosphonate (VP) at the 5′-end of the sense or antisensestrand.
 12. A pharmaceutical composition comprising the dsRNA agentaccording to claim 1 in combination with a pharmaceutically acceptablecarrier or excipient.
 13. A method for inhibiting the expression of atarget gene comprising the step of administering the dsRNA agentaccording to claim 1, in an amount sufficient to inhibit expression ofthe target gene.
 14. The method of claim 13, wherein the dsRNA agent isadministered through subcutaneous or intravenous administration.
 15. Amethod for delivering polynucleotide to specific target in a subject byadministering the dsRNA agent according to claim
 1. 16. The method ofclaim 15, wherein said administering step is carried out by anadministration means comprising intramuscular, intrabronchial,intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous,subcutaneous, cerebrospinal, or combinations thereof.
 17. A method fordelivering a polynucleotide to specific target of a subject, the methodcomprising: delivering a dsRNA agent according to claim 1 bysubcutaneous administration into the subject, such that thepolynucleotide is delivered into specific target of the subject.
 18. ThedsRNA agent of claim 1, wherein n⁴ is
 0. 19. The dsRNA agent of claim 1,wherein each B1, B2, and B3 is independently 2′-OMe or 2′-F.
 20. ThedsRNA agent of claim 1, wherein C1 is at position 14-17 of the 5′-end ofthe sense strand, n⁴ is 1, and C1 is a glycol nucleic acid (GNA). 21.The dsRNA agent of claim 1, wherein C1 is at position 14-17 of the5′-end of the sense strand, n⁴ is 1, C1 is a glycol nucleic acid (GNA),and each B1, B2, and B3 is independently 2′-OMe or 2′-F.
 22. The dsRNAagent of claim 1, wherein n⁴ is 0, and each B1, B2, and B3 isindependently 2′-OMe or 2′-F.
 23. The dsRNA agent of claim 1, whereineach B1 is independently 2′-OMe or 2′-F, and each B2 and B3 is 2′-OMe.24. The dsRNA agent of claim 1, wherein each B2 and B3 is 2′-OMe.